Biocompatibility and interaction of porous alumina-zirconia scaffolds with adipose-derived mesenchymal stem cells for bone tissue regeneration.

Replacement of bone defects with bone graft or implant is an important therapeutic strategy that has been used in routine practice. However, the identification of biomaterials that can mimic natural bone properties and serve as bone substitutes remains a major challenge. In this context, alumina-zirconia (Al2O3/ZrO2) nanocomposites emerge as potential alternatives for biomedical applications, owing to their high mechanical strength, wear resistance, and biocompatibility. In this sense, in this study, we prepared porous Al2O3/ZrO2 nanocomposites (scaffolds) using the gelcasting method and biomimetically coated them with calcium phosphate (CaP). We evaluated the biocompatibility of the scaffolds using the quantitative MTT cytotoxicity test in L929 cells. Moreover, rabbit adipose-derived mesenchymal stem cells (rADMSCs) were seeded with CaP-containing and CaP-free porous samples to evaluate cell proliferation and cell-scaffold interaction in vitro. Our results showed that the Al2O3/ZrO2 scaffolds were non-cytotoxic, and there were no significant differences between CaP-containing and CaP-free scaffolds in terms of cell growth and adhesion. In contrast, when co-cultured with rADMSCs, the scaffolds enhanced cell proliferation and cell adhesion. The rADMSCs adhered and migrated through the pores of the scaffold and anchored to different poles with differentiated elongated structures. These results suggest osteogenic differentiation of rADMSCs in response to mechanical loading of Al2O3/ZrO2 scaffolds. Therefore, we conclude that Al2O3/ZrO2 scaffolds have demonstrated significant implications in bone tissue engineering and are valuable biomaterials for bone replacement.

Protocol to Study the Role of Extracellular Vesicles During Induced Stem Cell Differentiation.

Extracellular vesicles (EVs) are vesicles released by cells, which due to their cargo and cell membrane proteins induce changes in the recipient cells. These vesicles can be a novel option to induce stem cell differentiation. Here we described a method to induce mesenchymal stem cell differentiation (MSC) into neuron-like cells using small EVs from neurons. First, we will describe a method based on neurons to induce adipocyte derived stem cells differentiation, a type of MSC, by coculturing both using inserts. Secondly, we will describe a follow-up method by using only isolated neuron-derived small EVs to directly induce ADSC differentiation in neuron-like cells. Importantly, in both methods it is possible to avoid the direct cell-to-cell contact, thus allowing for the study of soluble factors role during stem cell differentiation.

The Use of Bone Marrow-Derived Stem Cells in the Treatment of a Comminuted Tibiotarsal Fracture in a Juvenile Red-legged Seriema (Cariama cristata).

An adult red-legged seriema (Cariama cristata) presented with a comminuted fracture of the tibiotarsus and fibula. Surgery was performed, and a type II external fixator, with 2 distal and 2 proximal pins, was used to stabilize the fracture. After a 10-day stabilization period, the bird developed a second fracture on the same bone, proximal to the first fracture site. Another surgery was performed on the seriema similar to the first one. However, in this second surgical procedure a single pin, instead of 2 perpendicular pins, was placed proximally to the fracture site. After the second surgical procedure, bone marrow stem cells (BMSCs) from the seriema's left ulna were collected. Twenty-seven days after the second surgery, the BMSCs were transplanted, into the fracture sites. Twenty-four days after the stem cells were injected into the fractures (51 days after the second surgical procedure), radiographic images revealed healing bone calluses at the fracture sites. The fracture healing was relatively long for this case (a total of 75 days). The addition of bone marrow stem cell therapy to the use of external fixation may have contributed to the healing observed radiographically 24 days after administration; therefore, bone marrow stem cell therapy, in addition to traditional surgical fracture reduction and stabilization, may be a promising therapeutic approach for avian cases with similar injuries and bone anatomy. However, as this is a single case, this therapeutic modality deserves further application and study. Moreover, we suggest modifications in the bone marrow stem cell collection and therapy, which may be useful for future studies and application involving birds.

Characterization and Immunomodulation of Canine Amniotic Membrane Stem Cells.

PURPOSE: Amniotic membrane stem cells have a high capacity of proliferation, cell expansion, and plasticity, as well as immunomodulatory properties that contribute to maternal-fetal tolerance. Owing to the lack of research on human amniotic membrane at different gestational stages, the canine model is considered ideal because of its genetic and physiological similarities. We aimed to characterize the canine amniotic membrane (CAM) cell lineage in different gestational stages and evaluate the expression of immunomodulatory genes. MATERIALS AND METHODS: Twenty CAMs from early (20-30 days) (n=7), mid- (31-45 days) (n=7), and late gestation (46-63 days) (n=6) stages were studied. The cell features were assessed by cell viability tests, growth curve, colony-forming units, in vitro differentiation, cell labeling for different immunophenotypes, and pluripotent potential markers. The cells were subjected to RT-PCR and qPCR analysis to determine the expression of IDO, HGF, EGF, PGE2, and IL-10 genes. RESULTS: CAM cells exhibited a fibroblastoid morphology and adherence to plastic with an average cell viability of 78.5%. The growth curve indicated a growth peak in the second passage and we obtained an average of 138.2 colonies. Osteogenic, chondrogenic, and adipogenic lineages were confirmed by in vitro differentiation assays. Cellular immunophenotyping experiments confirmed the presence of positive mesenchymal markers (CD90 and CD105) and the low or negative expression of hematopoietic markers (CD45 and CD34). Qualitative analysis of the immunomodulatory functions indicated the expression of the IDO, HGF, EGF5, and PGE2 genes. When stimulated by interferon-gamma, CAM cells exhibited higher IDO levels throughout gestation. CONCLUSION: The CAMs from different gestational stages presented features consistent with mesenchymal stem cell lineage; better results were observed during the late gestation stage. Therefore, the gestational stage is a key factor that may influence the functionality of therapies when using fetal membrane tissues from different periods of pregnancy.

An improved method for studying mouse diaphragm function.

Dysfunction in the contractile properties of the diaphragm muscle contributes to the morbidity and mortality in many neuromuscular and respiratory diseases. Methods that can accurately quantify diaphragm function in mouse models are essential for preclinical studies. Diaphragm function is usually measured using the diaphragm strip. Two methods have been used to attach the diaphragm strip to the force transducer. The suture method is easy to adopt but it cannot maintain the physiological orientation of the muscle fibers. Hence, results may not accurately reflect diaphragm contractility. The clamp method can better maintain diaphragm muscle fiber orientation but is used less often because detailed information on clamp fabrication and application has never been published. Importantly, a side-by-side comparison of the two methods is lacking. To address these questions, we engineered diaphragm clamps using mechanically highly durable material. Here, we present a detailed and ready-to-use protocol on the design and manufacture of diaphragm clamps. Also, we present a step by step protocol on how to mount the diaphragm strip to the clamp and then to the muscle force measurement system. We compared the diaphragm force from the same mouse with both suture and clamp methods. We found the clamp method yielded a significantly higher muscle force. Finally, we validated the utility of the clamp method in the mdx model of Duchenne muscular dystrophy. In summary, the clamp method described in this paper yields reliable and consistent diaphragm force data. This method will be useful to any laboratory interested in performing mouse diaphragm function assay.

Morphological and Molecular Analysis of In Vitro Tubular Structures from Bovine Yolk Sac-Derived MSCs.

The yolk sac is an extraembryonic membrane, of saccular form, connected to the ventral region of the embryo. It is the main source of nutrition for the embryo during the period when the placenta is not fully formed. The aim of this study was to generate tubular structures using mesenchymal stem cells from the bovine yolk sac (bYS-MSCs) and determine if these structures can be a model for in vitro vasculogenesis. The evaluation of this tissue by histochemistry revealed a strong marking of collagen fibers and PAS technique negativity. In transmission electron microscopy, cytoplasmic organelles with large nuclei were observed. The vessel formation assay on a Matrigel substrate showed that the mesenchymal cells of the yolk sac without growth factors (VEGF) are capable of forming branches, sprouting cells, and tubular structures similar to capillary blood. These tubular structures were xenotransplanted subcutaneously into the mesentery of BALB/c/nude mice; after 45 days, vascularized tissue and extensions of blood vessels around the tubular structures could be observed. Real-time PCR (qPCR) demonstrated an expression of the VEGF gene in different gestational age groups. No difference in distribution or expression was detected among groups. Our results suggest that the spontaneous formation of tubules from the yolk sac can be an experimental model to elucidate initial organogenesis and the possible formation of blood capillaries from in vitro mesenchymal cells and possible route of organoid production.

Evaluation of the applicability of musculoskeletal ultrasonography of the thoracolumbar and lumbar spine segment of healthy dogs / Avaliação da aplicabilidade da ultrassonografia musculoesquelética do segmento toracolombar e lombar da coluna vertebral de cães hígidos

Changes in the spine of dogs are usually detected in clinical and in surgical practice. Few studies exist on musculoskeletal ultrasound anatomy of the thoracolumbar and lumbar segments of the normal spine of dogs. This study aimed to compare the normal musculoskeletal ultrasound anatomy of the T10-S1 vertebral segments with images obtained with magnetic resonance imaging (MRI), computed tomography (CT), and anatomical structures, and to establish the ability to identify structures using these modalities. Ultrasound scans allowed visualization of the muscles of the region, articular processes, spinous process, interspinous ligament, and yellow ligament in the lumbosacral window. Computed tomography images provided better bone details, compared to ultrasound images. Low-field MRI allowed the identification of the same structures identified with ultrasound imaging, and allowed the identification of cerebrospinal fluid, transverse processes, and provided improved detail of the intervertebral discs and spinal cord. Knowledge of ultrasound anatomy of the region may allow the the identification of muscle and ligament injuries. Thus, in cities where CT and MRI are inaccessible, ultrasonography of the region could be a good alternative to identify possible changes not observable with radiographic examination or to complement radiographic examination.(AU)

Alterações na coluna vertebral de cães são comumente encontradas na rotina clínica e cirúrgica veterinária. Existem poucos estudos sobre a anatomia ultrassonográfica musculoesquelética do segmento toracolombar e lombar da coluna vertebral normal de cães. O objetivo deste trabalho foi comparar a anatomia ultrassonográfica musculoesquelética normal dos segmentos vertebrais T10-S1 com imagens obtidas pela ressonância magnética, tomografia computadorizada e peças anatômicas visando demonstrar a sua capacidade de identificação de estruturas. A varredura ultrassonográfica permitiu a visibilização da musculatura da região, processos articulares, processos espinhosos, ligamentos interespinhosos e ligamento amarelo na janela lombossacra. A tomografia computadorizada forneceu imagens com melhor detalhamento ósseo quando comparada ao exame ultrassonográfico. A ressonância magnética de baixo campo permitiu a identificação das mesmas estruturas que o exame ultrassonográfico acrescido da identificação do líquido cerebroespinal, processos transversos e melhor detalhamento dos discos intervertebrais e medula espinhal. Com o conhecimento da anatomia ultrassonográfica da região, acredita-se que lesões musculares e ligamentares possam ser identificadas. Vale salientar que em cidades onde a tomografia computadorizada e a ressonância magnética não estejam acessíveis a ultrassonografia da região pode ser uma boa alternativa para identificar possíveis alterações não visibilizadas ao exame radiográfico, ou complementá-lo.(AU)

Assessment of the vertebral heart scale and cardiothoracic ratio to standardize the heart size of collared peccaries (Tayassu tajacu Linnaeus, 1758) restrained with ketamine and midazolam / Avaliação do vertebral heart scale e índice cardiotorácico na padronização do tamanho cardíaco de cateto (Tayassu tajacu Linnaeus, 1758) contidos com cetamina e midazolam

Wild species, especially those threatened with extinction, are increasingly being investigated to obtain information that can be useful for their preservation. The objective of the present study was to standardize the vertebral heart scale (VHS) and cardiothoracic ratio (CTR) of the collared peccary (Tayassu tajacu Linnaeus, 1758) sedated with ketamine and midazolam. Fourteen clinically healthy collared peccaries were examined in the two-year age group weighing 15-22kg. The animals were submitted to digital radiography of the thorax in lateral and dorsal ventral projections to calculate the VHS and CTR. The VHS mean values for males and females was 8.88±0.51v for right recumbency and 8.84±0.39v for left decubitus, and there were no significant between-gender differences regarding recumbency (p>0.05). The CTR showed mean values of 0.50±0.05 (males) and 0.45±0.04 (females), but the gender-differences were not significant (p>0.05). A positive correlation was shown between VHS and CTR (r=0.98, right decubitus; r=0.96, left decubitus). Establishing reference values for heart measurements in collared peccaries using digital radiography of the thorax permitted standardization of the VHS and CTR values for this wild species. In the studied wild animal model, the VHS and CTR heart assessment indexes were shown to be essential diagnostic tools for investigations of alterations in the size of the cardiac silhouette.(AU)

Espécies silvestres têm sido cada vez mais exploradas como forma de obter informações que favoreçam sua conservação, especialmente aquelas ameaçadas de extinção. Este trabalho teve por objetivo a padronização dos valores de VHS (vertebral heart scale) e índice cardiotorácico (ICT) de catetos (Tayassu tajacu Linnaeus, 1758) contidos com Cetamina e Midazolam. Foram avaliados 14 catetos clinicamente saudáveis, com faixa etária de 2 anos e variação média de peso entre 15 a 22Kg. Os animais foram submetidos a radiografia digital de tórax em projeções laterolaterais e dorsoventrais para o cálculo do vertebral heart scale (VHS) e Índice Cardiotorácico (ICT). O VHS evidenciou valores médios entre machos e fêmeas de 8,88±0,51v para decúbito direito e 8,84±0,39v para decúbito esquerdo, não ocorrendo diferença estatística entre os decúbitos (p>0,05). O ICT revelou valores médios de 0,50±0,05 (machos) e de 0,45±0,04 (fêmeas), não sendo verificada diferença estatística significativa entre os sexos (p>0,05). Verificou-se correlação positiva entre VHS e o ICT (r=0,98, decúbito direito, r= 0,96, decúbito esquerdo). O estabelecimento dos valores de referência para mensurações cardíacas em catetos, a partir de radiografias digitais do tórax, permitiu a padronização do VHS e ICT para esta espécie silvestre. No modelo animal silvestre estudado, os índices de avaliação cardíaca VHS e ICT mostraram-se como uma ferramenta diagnóstica imprescindível para investigações sobre as alterações do tamanho da silhueta cardíaca.(AU)

Abdominal B-mode and Doppler ultrasonography of chemically restrained agouti (Dasyprocta prymnolopha Wagler, 1831) / Ultrassonografia abdominal em Modo B e Doppler de cutias (Dasyprocta prymnolopha Wagler, 1831) contidas quimicamente

Agoutis are small-sized wild animals whose body weight can reach up to 4kg, and are found throughout Brazil. They are considered important seed dispersers, especially for big trees and there are species that rely almost exclusively on these animals for their territorial distribution. The objective of the present study was B scan and Doppler ultrasound characterization of the abdominal organs of healthy agoutis reared in captivity. Fifteen agoutis, chemically restrained, were used from the Nucleus for Wild Animal Studies and Conservation (Núcleo de Estudos e Preservação de Animais Silvestres - NEPAS), CCA-UFPI, submitted to B scan and Doppler ultrasound examination. The urinary bladder wall was hyperechogenic, thin, smooth and regular throughout its anatomic path, with 0.09±0.03cm mean thickness. The kidneys showed fine and homogeneous echotexture, preserved global echogenicity, hyperechogenic in relation to the spleen and isoechogenic or discreetly hyperechogenic in relation to the liver. The spectral Doppler trace showed systolic and diastolic peaks, wide and thread-like, with low flow resistance and a continuous and full diastolic portion that decreased gradually during the diastole (75.83±1.42cm/s, for the right kidney and 80.43±1.22cm/s, for the left kidney). The right adrenal gland was 0.61-1.18cm long and 0.17-0.32cm in diameter, while the left adrenal gland was 0.62-1.16 long with 0.14-0.25cm diameter. The agouti spleen was filiform in shape, with pointed poles and 1.02±0.18cm in diameter. The agouti liver occupied all the abdominal cavity cranial space in direct contact with the diaphragm. The intrahepatic vascular flow allowed individualization of the portal vein (PV) and hepatic vein (HV). The portal veins were distinguished from the hepatic veins mainly by their wall echogenic pattern. The pancreas was 0.51±0.1 cm thick and the pancreatic duct measured 0.12±0.02cm. The stomach was placed to the left the spleen and to the right of the proximal intestine and the transversal colon and the walls were 0.16±0.05cm thick. The abdominal aorta was 0.43±0.04cm in diameter and showed 95.2±2.16cm/s vascular flow. This study characterized agouti organs and abdominal blood vessels by B scan and Doppler ultrasound, that permitted definition of the size, shape, position, echogenicity and echotexture of the anatomic constituents and established reference values for the vascular network and blood flow in the species.(AU)

As cutias são animais silvestres de pequeno porte, cujo peso corpóreo pode chegar até 4kg, e existem em todo território brasileiro. São considerados importantes dispersores de sementes, especialmente para árvores de grande porte, existindo espécies que dependem quase que exclusivamente destas para sua distribuição territorial. Este trabalho teve por objetivo a caracterização ultrassonográfica modo B e Doppler dos órgãos abdominais de cutias hígidas criadas em cativeiro. Foram utilizadas 15 cutias, contidas quimicamente, oriundas do Núcleo de Estudos e Preservação de Animais Silvestres - NEPAS, CCA-UFPI, submetidas a exame ultrassonográfico em modo B e Doppler. A parede da vesícula urinária presentou-se hiperecogênica, fina, lisa e regular em todo seu trajeto anatômico, com espessura média de 0,09±0,03cm. Os rins demonstraram ecotextura fina e homogênea, ecogenicidade global preservada, hipoecogênico em relação ao baço e isoecogênico ou discretamente hipoecogênico em relação ao fígado. O traçado em Doppler espectral mostrou pico sistólico e diastólico, amplo e afilado, exibindo baixa resistência de fluxo, com uma porção diastólica contínua e cheia, que diminui gradativamente no decorrer da diástole (75,83±1,42cm/s para o rim direito e 80,43±1,22 cm/s para o esquerdo. A adrenal direita apresentou uma variação de comprimento entre 0,61 a 1,18cm e diâmetro variando entre 0,17 a 0,32cm, enquanto a adrenal esquerda evidenciou comprimento de 0,62 a 1,16 e diâmetro de 0,14 a 0,25cm. O baço das cutias mostrou formato filiforme, com polos pontiagudos e diâmetro de 1,02±0,18cm. O fígado da cutia ocupa todo o espaço cranial da cavidade abdominal, em contato direto com o diafragma. O fluxo vascular intrahepático permitiu individualizar as veias porta (VP) e veias hepáticas (VH). As veias porta foram distinguidas, particularmente pelo padrão ecogênico de suas paredes, quando comparadas com as veias hepáticas. A espessura do pâncreas foi de 0,51±0,1cm e o ducto pancreático mediu 0,12±0,02cm. O estômago relaciona-se à esquerda com o baço e a direita com o duodeno proximal e colón transverso. Sua espessura de parede mensurada foi de 0,16±0,05cm. A aorta abdominal possui diâmetro de 0,43±0,04cm e fluxo vascular de 95,2±2,16cm/s. Este estudo caracterizou os órgãos e vasos sanguíneos abdominais de cutias, por meio de ultrassonografia modo B e Doppler, o que permitiu definir o tamanho, formato, posição, ecogenicidade, ecotextura dos constituintes anatômicos, além de estabelecer valores de referência para a rede vascular e fluxo sanguíneo na espécie.(AU)

Dynamics of male canine germ cell development.

Primordial germ cells (PGCs) are precursors of gametes that can generate new individuals throughout life in both males and females. Additionally, PGCs have been shown to differentiate into embryonic germ cells (EGCs) after in vitro culture. Most studies investigating germinative cells have been performed in rodents and humans but not dogs (Canis lupus familiaris). Here, we elucidated the dynamics of the expression of pluripotent (POU5F1 and NANOG), germline (DDX4, DAZL and DPPA3), and epigenetic (5mC, 5hmC, H3K27me3 and H3K9me2) markers that are important for the development of male canine germ cells during the early (22-30 days post-fertilization (dpf)), middle (35-40 dpf) and late (45-50 dpf) gestational periods. We performed sex genotype characterization, immunofluorescence, immunohistochemistry, and quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) analyses. Furthermore, in a preliminary study, we evaluated the capacity of canine embryo PGCs (30 dpf) to differentiate into EGCs. To confirm the canine EGCs phenotype, we performed alkaline phosphatase detection, immunohistochemistry, electron and transmission scanning microscopy and RT-qPCR analyses. The PGCs were positive for POU5F1 and H3K27me3 during all assessed developmental periods, including all periods between the gonadal tissue stage and foetal testes development. The number of NANOG, DDX4, DAZL, DPPA3 and 5mC-positive cells increased along with the developing cords from 35-50 dpf. Moreover, our results demonstrate the feasibility of inducing canine PGCs into putative EGCs that present pluripotent markers, such as POU5F1 and the NANOG gene, and exhibit reduced expression of germinative genes and increased expression of H3K27me3. This study provides new insight into male germ cell development mechanisms in dogs.

In vitro identification of a stem cell population from canine hair follicle bulge region.

Skin is an extensive and easily accessible organ possessing various cell types that are constantly renewed. Previous studies have suggested the presence of a stem cell niche at the bulge region of the hair follicle, which contains cells positive for CD200 and CD34. Thus, this study sought to identify these cell populations in canine skin cells using the following methods 1- collecting samples of adult and fetal skin and isolating and culturing these cells using a method of simple enzymatic digestion and 2- testing the cell cultures for CD200 and CD34 in vitro and comparing them with skin tissue samples (in situ). Immunofluorescence results were negative for both CD200 and CD34 in frozen and paraffin embedded tissue, whereas the analysis showed that cultured cells positive for CD34, CD200 and double positive cells could be visualized in different percentages. Additionally, the pluripotency marker OCT4 was positive in the isolated cells. Analysis of CD34, CD200 and OCT4 by RT-qPCR showed that there is expression in fetal and adult cells, although no difference was observed between groups. Our results suggest that bulge stem cells from both fetuses and adult dogs were reported with the use of CD34 and CD200 markers in this study, and further techniques for cell isolation and in vitro cultivation are needed in order to obtain enriched populations of skin stem cells in dogs.

Biometric, B-mode and color Doppler ultrasound assessment of eyes in healthy dogs / Avaliação biométrica, ultrassonográfica em modo-B e por Doppler colorido ocular de cães hígidos

B-scan ultrasonography is an important diagnostic tool that allows characterization of internal organ anatomy and, when complemented by Doppler ultrasound, allows vascular hemodynamic assessment, increasing the diagnostic accuracy. Thus, the aim of the present study was the B-scan ultrasound characterization and measurement of the eyeball segments and assessment of the external ophthalmic artery by color and pulsed Doppler. Sixty eyeballs were assessed from 30 dogs of different breeds using an 8.5MHz microconvex transductor. First, biometry was performed by B-scan of the following segments: axial length (M1), anterior chamber depth (M2), lens thickness (M3), lens length (M4), vitreous chamber depth (M5), optical disc length (M6) and optic nerve diameter (M7). Colored Doppler identified the external ophthalmic article and pulsed Doppler assessed its flow, and the following were measured: systolic peak velocity (VPS), final diastolic velocity (VDF), resistivity index (IR) and pulse index (IP). No statistical difference was observed for the biometric values of the eye segments between the right and left eyes (p>0.05). The vitreous chamber depth (M5) was shown to be the biometric variable with greatest bilateral symmetry, varying from 0.79 to 0.87cm and 0.78 to 0.86cm for the right and left eye, respectively. The ophthalmic artery was visualized over the optic nerve towards the eyeball, with flow stained red. There was no significant statistical difference between the Doppler velocimetric values for the ophthalmic artery between the right and left eye of the animals assessed (p>0.05). The mean resistivity index (RI) showed average values equal to 0.63±0.03, bilaterally. The mean base velocity was 17.50cm/s and 18.18cm/s at the systolic peak and 6.21cm/s and 6.68cm/s at the end of the diastole, for the right and left eyes respectively. The anatomic, biometric and hemodynamic characterization using the ultrasound B-scan and the Doppler modalities permitted the ultrasonographic and Doppler velocimetric assessment of the eyeball components in dogs of different breeds, and it can be used in ophthalmic clinical routine to identify eye pathologies.

O exame ultrassonográfico modo-B é uma importante ferramenta diagnóstica que permite caracterizar a anatomia interna dos órgãos e, complementada pelo exame modo Doppler, possibilita realizar a avaliação hemodinâmica vascular, aumentando a acurácia diagnóstica. Desta forma, este trabalho teve como objetivo a caracterização ultrassonográfica e mensuração dos segmentos do bulbo ocular modo-B, assim como a avaliação da artéria oftálmica externa pelo Doppler colorido e pulsado. Foram avaliados 60 bulbos oculares de 30 cães de diferentes raças utilizando transdutor microconvexo de 8,5MHz. Inicialmente foi realizada biometria por meio do exame em modo-B dos seguintes seguimentos: comprimento axial (M1), profundidade da câmara anterior (M2), espessura da lente (M3), comprimento da lente (M4), profundidade da câmara vítrea (M5), comprimento do disco óptico (M6) e diâmetro do nervo óptico (M7). A artéria oftálmica externa foi identificada pelo Doppler colorido e seu fluxo foi avaliado por meio do Doppler pulsado, sendo mensurados: velocidade do pico sistólico (VPS), velocidade diastólica final (VDF), índice de resistividade (IR) e índice de pulsatilidade (IP). Não foi verificada diferença estatística para os valores biométricos dos seguimentos oculares entre os olhos direito e esquerdo (p>0,05). A profundidade da câmera vítrea (M5), mostrou-se a variável biométrica com maior simetria bilateral, variando entre 0,79 a 0,87cm e 0,78 a 0,86cm para o olho direito e esquerdo, respectivamente. A artéria oftálmica foi visibilizada sobre o nervo óptico em direção ao bulbo ocular, com fluxo marcado em vermelho. Não houve diferença estatística significativa entre os valores Dopplervelocimétricos da artéria oftálmica entre do olho direito e esquerdo dos animais avaliados (p>0.05). O índice de resistividade (IR) médio evidenciou valores médios sendo igual a 0,63±0,03, bilateralmente. A velocidade basal média foi 17,50cm/s e 18,18cm/s no pico sistólico e 6,21cm/s e 6,68cm/s no final da diástole, para os olhos direito e esquerdo, respectivamente. A caracterização anatômica, biométrica e hemodinâmica utilizando o exame ultrassonográfico modo-B e as modalidades do Doppler permitiram a avaliação ultrassonográfica e Dopplervelocimétrica dos componentes do bulbo ocular nos cães de diferentes raças, podendo ser utilizados na rotina clínica oftalmológica para identificação de patologias oculares.

Abnormal carbohydrate metabolism in a canine model for muscular dystrophy.

The canine golden retriever muscular dystrophy (GRMD) model is the best animal model for studying Duchenne muscular dystrophy in humans. Considering the importance of glucose metabolism in the muscles, the existence of metabolic and endocrine alterations in a wide range of muscular dystrophies, and the pre-existing relationship between blood insulin concentration and muscular atrophy, the present study aimed to evaluate the postprandial glucose and insulin response in GRMD dogs. A total of eighteen golden retriever dogs were randomly distributed into three experimental groups: healthy/control (G1), female GRMD carriers (G2), and male dogs affected by GRMD (G3). Higher plasma resting glucose levels (P = 0·0047) were seen in G2 and G3 compared with G1, as was the case for minimum (P = <0·0001), mean (P = 0·0002) and maximum (P = 0·0359) glucose values for G3 compared with G1. Fructosamine concentrations were in accordance with reference values found in the literature for dogs. Insulin levels were lower in G3 compared with G1 (P = 0·0065); however, there was no evidence of insulin resistance according to the homeostasis model assessment index values obtained. As for the evaluation of postprandial responses, fluctuations of glucose (P = 0·0007) and insulin (P = 0·0149) were observed in G1 and G2, while in G3 the values remained constant. The results allowed us to identify metabolic changes related to carbohydrate metabolism in GRMD dogs, highlighting the importance of adequate food management for these animals.

Aspecto morfológico da interface entre o compósito, constituído de quitosana e polimetilmetacrilato, e a falha óssea de tíbia de coelhos / Morphological aspect of the interface between the composite, made of chitosan and polymethylmethacrylate, and bone failure of the rabbit tibia

As afecções ortopédicas com perda de tecido ósseo são um desafio não só na medicina veterinária mas também na medicina humana. Analisou a interface entre compósito, constituído de esferas de quitosana e polimetilmetacrilato em falha óssea (leito receptor) de tíbia de coelhos, por meio de técnica radiológica, avaliação macroscópica e pela microscopia eletrônica de varredura, em diferentes tempos. Foram utilizados 12 coelhos adultos da raça Nova Zelândia, divididos em quatro Grupos Experimentais (E1, n=3; E2, n=3; E3, n=3 e E4, n=3), que tiveram as falhas ósseas das tibiais direitas preenchidas com compósito, e avaliadas no pós-operatório imediato, aos 30, 60 e 90 e 120 dias. Dos compósitos implantados nas tíbias de coelhos, apenas dois permaneceram em seus leitos receptores, enquanto que os demais se encontravam encapsulados no tecido subcutâneo. As esferas de quitosana, presentes nas superfícies dos biomateriais implantados, que mantiveram contato direto com o leito receptor de tíbias de coelhos apresentavam-se preservadas e não integraram ao tecido ósseo. Diante disso, para melhor compreensão do comportamento da quitosana como substituto ósseo, novas pesquisas serão necessárias.(AU)

Orthopedic diseases with bone loss are challenging in both veterinary and human medicine. The aim of this investigation was to analyze and compare the reactions at the interface between the composite, made of chitosan and polymethylmethacrylate, and the bone defect (receptor site) of the rabbits tibia through radiological and microscopic techniques and by scanning electron microscopy, in different periods. Twelve adult New Zealand rabbits were divided into four experimental groups (E1, n=3; E2, n=3; E3, n=3 and E4, n=3), which had the right tibial bone defects filled with the composite, and evaluated in the immediate postoperative, 30, 60, 90 and 120 days. Composite implanted in the tibia of rabbits, only two remained in their beds receivers, while the remaining were encapsulated in the subcutaneous tissue. Spheres of chitosan present in the biomaterial that has been deployed and were in direct contact with the bone defect, were preserved, however, were not integrated into the bone tissue. Therefore, to understand the behavior of chitosan as a bone substitute, further research is needed.(AU)

Bioimpressão e produção de mini-órgãos com células tronco / Bioprinting with stem cells and production of mini-organs

A bioimpressão é considerada uma fonte promissora no desenvolvimento celular, e na produção de mini-órgãos, válulas, cartilagens que futuramente poderão ser utilizados na terapia para transplantes em animais e humanos. Assim, essa técnica poderá ser utilizada como uma terapia eletiva, no tratamento de injúrias e principalmente no tratamento de doenças crônico-degenerativas. Em humanos essa terapia está sendo pesquisada a fim de auxiliar a medicina no tratamento e regeneração de tecidos impressos a partir de arcabouços de células desenvolvidas a partir de células-tronco, biomateriais e impressões em 3D. O uso dessa tecnologia é também um auxiliar nas pesquisas oncológicas com o intuito de projetar e avaliar a proliferação celular de tumores, bem como a ação de novos medicamentos quimioterápicos. No entanto, a maior limitação para o uso da terapia utilizando-se a impressora de células, órgãos e tecidos em 3D é a falta de protocolos unificados com metodologias reprodutíveis e detalhadas; com o objetivo de viabilizar a utilização da impressora e a impressão de células, órgãos e tecidos em 3D. Dessa forma, esta revisão busca reunir as publicações mais atuais na área, as quais destacam os avanços no uso de bioimpressão com células-tronco, a fim de descrever as principais técnicas e os potenciais de utilização como alternativa terapêutica na medicina humana e veterinária.(AU)

The bioprinting is considered a promising source in cell development, and production of mini-organs, valves, cartilage that may eventually be used in therapy for transplantation in animals and humans. It can also be used as an elective therapy in the treatment of injuries and treatment of chronic degenerative diseases. In humans, this therapy is been studied mainly in the treatment and regeneration of tissues printed from scaffold cells developed from stem cells, biomaterials and impressions in 3D. This technology is also an aid for the study of the formation of tumors, in order to design and evaluate the cellular proliferation of the tumors and the action of new chemotherapy drugs. However, the main drawback to this therapy is the lack of standardized protocols with reproducible and detailed methodologies with the aim of enabling the use of bioprinting and printing cells, tissues and organs in 3D. Thus, this review seeks to bring together the most current publications of the bioprinting area in order to describe the technique and its potential use as a therapeutic alternative.(AU)

Morfologia da traqueia e lobação pulmonar de Leopardus pardalis (jaguatirica) / Morphology of the trachea and lung lobation of Leopardus pardalis (ocelot)

A espécie Leopardus pardalis, conhecida popularmente como jaguatirica, é um felino de médio porte com ampla distribuição em território nacional e está incluída na lista de espécies da fauna brasileira ameaçadas de extinção. Este estudo teve como objetivo caracterizar os aspectos macroscópicos e microscópico da traqueia e lobos pulmonares em L. pardalis. As análises foram feitas utilizando cinco exemplares de jaguatirica, sendo dois machos e três fêmeas, adultos, três desses exemplares foram a óbito por atropelamento em rodovias próximas ao município de Alta Floresta, e dois doados após a morte pelo IBAMA ao o Laboratório de Zoologia e Morfologia Animal. Nos espécimes a traqueia era um tubo flexível formado por pares de arcos cartilaginosos, apresentando epitélio pseudo-estratificado não ciliado no seu revestimento interno, e outros estruturas básicas deste órgão. O pulmão se mostrou como um órgão constituído por lobos separados por fissuras com um parênquima usual aos demais mamíferos descritos. No que diz respeito às caraterísticas anatômicas da traqueia e pulmão se mostraram semelhantes à de outros mamíferos, já nos aspectos microscópicos da traqueia apresentou certas peculiaridades diferindo dos demais trabalhos encontrados. No que tange o aspecto do parênquima pulmonar as características histológicas não variaram com as que estão descritas.(AU)

The species Leopardus pardalis, popularly known as ocelot, is a medium sized cat with wide distribution in the national territory and is included in the list of species of Brazilian fauna threatened with extinction. This study aimed to characterize the macroscopic and microscopic aspects of the trachea and lung lobes in L. pardalis. Analyses were performed using five copies of ocelot, two males and three females, adults, three of these specimens died from being run over on highways nearby the municipality of Alta Floresta, and two donated after death by IBAMA to the Laboratory of Zoology and Animal Morphology. In the trachea specimens was a flexible tube formed by pairs of cartilaginous arches, with pseudo-stratified epithelium ciliated not in its inner lining, and other basic structures of this body. Lung proved as a body made up of lobes separated by cracks with a usual parenchyma to the other described mammals. With respect to the anatomic features of the trachea and lung were similar to other mammals, as in the microscopic aspects of the trachea showed certain peculiarities found differing from the other jobs. Regarding the appearance of the lung parenchyma histological characteristics did not differ with those described.(AU)

Evaluation of potential changes in liver and lung tissue of rats in an ischemia-reperfusion injury model (modified pringle maneuver).

In surgical procedures involving the liver, such as transplantation, resection, and trauma, a temporary occlusion of hepatic vessels may be required. This study was designed to analyze the lesions promoted by ischemia and reperfusion injury of the hepatic pedicle, in the liver and lung, using histopathological and immunohistochemical techniques. In total, 39 Wistar rats were divided into four groups: control group (C n = 3) and ischemia groups subjected to 10, 20, and 30 minutes of hepatic pedicle clamping (I10, n = 12; I20, n = 12; I30, n = 12). Each ischemia group was subdivided into four subgroups of reperfusion (R15, n = 3; R30, n = 3; R60, n = 3; R120, n = 3), after 15, 30, 60, and 120 minutes of reperfusion, respectively. Significant differences were observed in the liver parenchyma (P < 0.05) between the values of microvesicles and hydropic degeneration at different times of ischemia and reperfusion. However, the values of vascular congestion, necrosis, and pyknotic nuclei showed no significant differences (P > 0.05). In the lung parenchyma, a significant difference was observed (P < 0.05) between the values of alveolar septal wall thickening and inflammatory infiltration at different times of ischemia and reperfusion. However, there was no significant difference (P < 0.05) between the values of vascular congestion, bronchial epithelial degeneration, interstitial edema, and hemorrhage. The positive immunoreactivity of caspase-3 protein in the liver parenchyma (indication of ongoing apoptosis), showed no significant differences (P > 0.05) at different times of ischemia and reperfusion. In the pulmonary parenchyma, the immunoreactivity was not specific, and was not quantified. This study demonstrated that the longer the duration of ischemia and reperfusion, the greater are the morphological lesions found in the hepatic and pulmonary parenchyma.

Computerized electrocardiogram in agoutis (Dasyprocta prymnolopha Wagler, 1831) anesthetized with ketamine and midazolam / Eletrocardiografia computadorizada em cutias (Dasyprocta prymnolopha Wagler, 1831) anestesiadas com cetamina e midazolam

An electrocardiogram is a test that assesses heart electrical activity and is applied more frequently in the veterinary care of wild animals. The present study aimed to define the electrocardiogram pattern of agoutis (Dasyprocta prymnolopha Wagler, 1831) anesthetized with ketamine and midazolam. Eighteen clinically healthy agoutis (D. prymnolopha) were used from the Nucleus for Wild Animal Studies and Conservation (NEPAS) of the Federal University of Piauí, Brazil. The animals were chemically restrained with 5% ketamine hydrochloride at a dose of 15mg/kg and midazolam at a dose of 1mg/kg by intramuscular injection. Electrocardiogram tests were carried out by a computerized method with the veterinary electrocardiogram [Acquisition Model for Computer (ECG - PC version Windows 95) Brazilian Electronic Technology (TEB) consisting of an electronic circuit externally connected to a notebook computer with ECGPC-VET (TEB) software installed on the hard disc. In analysing the EKG results, significant differences were observed for QRS complex duration, PR and QT intervals and for R wave millivoltage between the genders; but we observed a significant influence of weight despite the gender. In the present experiment, the anaesthetic protocol was shown to be well tolerated by the agoutis, and no arrhythmias occurred during the time the animals were monitored. The reference values obtained should be used to better understand the cardiac electrophysiology of the species and for its clinical and surgical management.(AU)

O eletrocardiograma computadorizado é um dos meios de diagnóstico utilizado para avaliação do coração e vem sendo cada vez mais presente na rotina veterinária. Este trabalho teve por objetivo definir o padrão eletrocardiográfico de cutias (D. prymnolopha) anestesiadas com cetamina e midazolam. Foram utilizadas 18 cutias clinicamente saudáveis, provenientes do Núcleo de Estudos e Preservação de Animais Silvestres (NEPAS) da Universidade Federal do Piauí. Os animais foram submetidos à contenção química com cloridrato de cetamina a 5% na dosagem de 15mg/kg associado ao midazolam, na dosagem de 1mg/kg, por via intramuscular. Os exames eletrocardiográficos foram realizados pelo método computadorizado, com o eletrocardiógrafo veterinário (Módulo de Aquisição de ECG Para Computador (ECG - PC versão Windows 95) Tecnologia Eletrônica Brasileira (TEB) composto por um circuito eletrônico ligado externamente a um notebook, e de um software instalado no disco rígido do computador. Os valores de duração do complexo QRS, intervalos PR e QT, comparados entre machos e fêmeas, apresentaram diferença significativa. Em milivoltagem a onda R foi o único parâmetro que apresentou diferença significativa entre machos e fêmeas. O peso dos animais também foi significativamente diferente entre os gêneros. O protocolo anestésico mostrou-se bem tolerado pelos animais deste experimento, não ocorrendo quadros de arritmias durante o tempo de monitoramento dos animais.(AU)

Labeling of adipose-derived stem cells with quantum dots provides stable and long-term fluorescent signal for ex vivo cell tracking.

Stem cells derived from adipose tissue (ADSC) have been used in cell therapy as an alternative to treat chronic and degenerative diseases. Using biomedical and image trials to track the cells when infused in the target tissue is essential to control cell migration and adhesion. The objective of the present study was to label and assess the adhesion of goat adipose tissue-derived stem cells (g-ADSC) after cell infusion in animal models by tracking luminescent intracytoplasmatic nanocrystals. The cells were labeled by using Qdots. The g-ADSCs infused with nanocrystal were prepared either fresh or fixed and further visualized under a fluorescence microscope. The labeled cells were infused in the goat mammary glands and mouse testicles and kidneys via tail vein injection. Thirty days after cell infusion, biopsy was carried out for analyses. The g-ADSC cultures were presented with high cellularity and fibroblast morphology, even after infusion of the nanocrystals. It was possible, by processing in paraffin and under fluorescence microscopy, demonstrating the success of the labeling in the long term. Freezing mammary gland biopsies in liquid NO2 did not alter the quality of labeling with Qdots. Therefore, g-ADSCs can be labeled with intracytoplasmatic nanocrystals (Qdots) enabling their in vitro and ex vivo tracking.

Estudo comparativo da resistência mecânica da força de compressão entre biomateriais naturais, sintéticos e mistos / Comparative study of mechanical strength of compression between natural, synthetic and mixed biomaterials

As afecções ortopédicas com perda de tecido ósseo são um desafio tanto na medicina veterinária quanto na medicina humana. Portanto, não é raro ortopedistas se depararem com fraturas cominutivas irredutíveis de ossos longos, neoplasias ósseas ou não-uniões, que necessitam de procedimentos cirúrgicos reparadores, por meio da substituição de segmento ou preenchimento de falha óssea com o uso de biomateriais. Pretende-se com esta pesquisa avaliar e comparar a resistência mecânica entre biomateriais naturais, sintéticos e mistos. Foram utilizados sete grupos experimentais compostos por seis corpos de provas cada: Grupo 1 , tecido ósseo cortical de coelho (OSSO - controle); Grupo 2, cimento ósseo (CO); Grupo 3, cimento ósseo autoclavado (COA); Grupo 4, cimento ósseo e macrofragmento ósseo cortical de cão (COMaFO); Grupo 5, cimento ósseo e macrofragmento ósseo autoclavado de cortical de cão (COMaFOA); Grupo 6, cimento ósseo e microfragmento ósseo cortical de cão (COMiFO) e Grupo 7, cimento ósseo e microfragmento ósseo cortical de cão (COMiFOA). Os corpos de prova foram submetidos a ensaios mecânicos de compressão axial controlada em máquina universal de ensaio Emic®. O teste era interrompido quando ocorria queda brusca na curva do gráfico indicando falência da amostra. Em relação à Força máxima, os grupos COA, COMaFOA e COMiFOA não diferiram estatisticamente do grupo controle (OSSO; p>0,01). Já os grupos CO, COMaFO e COMiFO diferiram estatisticamente do grupo controle (OSSO; p<0,01). Quanto a rigidez relativa, os grupos COMaFOA e COMiFOA não diferiram estatisticamente do grupo controle (OSSO; p>0,01). Já os grupos CO, COA, COMaFO e COMiFO diferiram estatisticamente do grupo controle (OSSO; p<0,01). Comparando a deformação, os grupos COMaFo, COMaFOA e COMiFO não diferiram estatisticamente do grupo controle (OSSO; p>0,01). Já os grupos CO, COA e COMiFOA diferiram estatisticamente do grupo controle (OSSO; p<0,01). Conclui-se que apenas os grupos COMaFOA e COMiFOA apresentaram propriedades mecânicas muito semelhantes às do grupo controle (OSSO). Por isso, devido a essas características, esses dois biomateriais (COMaFOA e COMiFOA) seriam os mais indicados como substitutos na reparação de falhas ósseas.(AU)

The orthopedic diseases with bone loss are the challenge in both veterinary and human medicine. Therefore, the orthopedist commonly deal with irreducible comminuted fractures of long bones, bone tumors or non-unions, which require repairers surgical procedures, through the segment replacement or bone defect filling with biomaterials. The aim of this research is to evaluate and compare the mechanical strength of natural, synthetic and mixed biomaterials. Seven experimental groups of six test samples each were used: Group 1 rabbit cortical bone (BONE - control); Group 2, bone cement (CO); Group 3, bone cement autoclaved (COA); Group 4, bone cement and dog cortical bone macrofragment (COMaFO); Group 5, bone cement and bone autoclaved macrofragment dog cortical (COMaFOA); Group 6, bone cement and dog cortical bone microfragment (COMIFO) and Group 7, bone cement and dog cortical bone microfragment (COMiFOA). The specimens were subjected to axial compression mechanical tests controlled universal testing machine EMIC®. The test was stopped when there was sharp decline in the graph curve indicating failure of the sample. In relation to the maximum force, the COA groups COMaFOA and COMiFOA not statistically different from the control group (BONE; p> 0.01). Already the CO groups, COMaFO and COMIFO difeririram statistically the control group (BONE; p <0.01). The relative rigidity, the COMaFOA and COMiFOA groups did not differ statistically from the control group (BONE; p>0.01). Already the CO groups, COA, COMaFO and COMIFO differed significantly from the control group (BONE; p<0.01). Comparing the deformation, the COMaFo groups COMaFOA and COMIFO not statistically different from the control group (BONE; p>0.01). Already the CO groups, COA and COMiFOA differed significantly from the control group (BONE; p<0.01). It is concluded that only COMaFOA and COMiFOA groups showed very similar mechanical properties to the control group (BONE). Therefore, due to these characteristics, these two biomaterials (COMaFOA e COMiFOA) would be the most suitable as a substitute in the repair of bone defects.(AU)