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Neoplastic masses were evaluated in the rostral region of the mandible of three young adult cattle. In all three cases, the masses were macroscopically large, firm, ulcerated, infiltrative, whitish and solid, and led to tooth displacement and loss. Radiographically, the neoplastic masses were intraosseous and radiolucent with foci of radiopacity. Loss of radiopacity due to bone necrosis was seen in the mandibular bone adjacent to the neoplasms. Histologically, well-differentiated, infiltrative non-encapsulated mesenchymal neoplastic proliferation replaced the mandibular bone and extended to the oral mucosa in all three cases. The neoplastic cells had scant cytoplasm and fusiform or oval hyperchromatic nuclei with loose chromatin, and were arranged in bundles in various directions. Within the neoplastic tissue, there were mineralized bone trabeculae and unmineralized osteoid, lined by a layer of osteoblasts and osteocytes within the lacunae. The pre-existing bone tissue adjacent to the neoplasm had areas of necrosis and osteoclasis of variable extent and intensity. Based on the macroscopic, radiographic and microscopic findings, a diagnosis of mandibular ossifying fibroma was established in all three cattle.

Thirteen female Wistar rats were divided into two groups: one treated with ethanol and the other of untreated. Four newborns from each mother were selected and weighed, measured, and evaluated for physical characteristics. From these neonates, chondrocytes were extracted from the articular cartilages of the femur and tibia, and cultivated in a chondrogenic medium at 37oC and 5% CO2. At 7, 14, and 21 days of cultivation, alkaline phosphatase activity tests, MTT conversion to formazan, and percentage area covered by cells per field were performed. At 21 days, the percentage of PAS+ areas in 3D cultures was performed, as well as the evaluation of gene transcript expression for aggrecan, SOX-9, collagen type II, collagen X, Runx-2, and VEGF by real-time RT-PCR. The means were compared by Student’s t-test. The weight of the ethanol group neonates was significantly lower than that of the controls. Chondrocyte cultures from the ethanol group showed significantly higher AP activity, MTT conversion, and cell percentage. There was higher expression of collagen type II and lower expression of SOX-9 in the ethanol group. There was no difference in the percentage of PAS+ areas in pellets and in expression of aggrecan, collagen X, Runx-2, or VEGF between groups. In conclusion, prenatal exposure to ethanol alters the phenotype and activity of offspring chondrocytes, which may be mechanisms by which endochondral bone formation is compromised by maternal ethanol consumption.

This study aimed to evaluate the effect of maternal ethanol consumption during gestation and lactation on bone mass and osteogenic differentiation of mesenchymal stem cells of the bone marrow (BMMSCs) in rats. Thirteen adult Wistar rats were used. The rats were mated, and after confirmation of gestation, (day 0) they were distributed in two groups: the control group and the ethanol-treated group. From the ninth day of gestation, the rats of the ethanol and control groups were administered 40% alcoholic solution (4 g ethanol/kg) and distilled water, respectively, daily via gavage until the thirtieth day of lactation. The BMMSCs were extracted from the right femurs and tibiae and cultured using an osteogenic medium for 7, 14, and 21 days. The conversion of MTT to formazan crystals, alkaline phosphatase activity, and percentages of cells per field were analyzed. The number of mineralized nodules per field was examined, and quantification of the gene transcripts for osteopontin, osteocalcin, and BMP-2 was evaluated on day 21 by real-time RT-PCR. Morphometric evaluations of the percentage of trabecular bone and cortical thickness in the left femur and tibia were performed. The means were compared by the Student's t-test, and the differences were considered significant if p 0.05. The BMMSCs of the rats that consumed ethanol during gestation and lactation, when subjected to osteogenic differentiation in vitro, demonstrated higher conversion of MTT to formazan, higher alkaline phosphatase activity, a higher percentage of cells per field, higher expression of BMP-2, and higher synthesis of mineralized nodules when compared to those of control rat cells. However, there was no significant difference in the percentage of trabecular bone or cortical thickness between both groups. Hence, the consumption of ethanol during pregnancy and lactation did not alter the trabecular and cortical bone tissues of the femur and tibia compared with that of pregnant and lactating control rats that did not consume alcohol, despite BMMSCs showing higher osteogenic differentiation under in vitro conditions.

Summary Histopathological changes in tooth structures in dogs with calculus have not been described. The aim of this study was to evaluate the frequency of various histopathological changes in the dentine of teeth that had been surgically extracted from dogs with calculus. Data including breed, sex, age, reason for tooth extraction and dental history were obtained for each animal. A total of 158 teeth (45 incisors, 31 canines, 35 premolars and 47 molars) with calculus were extracted from 74 dogs of various ages and breeds. The teeth were decalcified, processed in paraffin wax and stained with haematoxylin and eosin for histopathological analysis. Of the 158 analysed teeth, 71 had dentinal changes, including 45 with external resorption cavities, 11 with osteodentine, six with internal resorption cavities, four with tertiary dentine, four with dentinal degeneration or fragmentation, and one with predentine degeneration or fragmentation. Canine teeth were the least commonly affected. Areas of dentinal degeneration or fragmentation unrelated to resorption cavities were only seen in the incisor and molar teeth. Dentinal changes and their frequencies were similar among the incisor, premolar and molar teeth. The presence or extension of dental calculus was not associated with the type or frequency of dentinal changes.

In forensic toxicology, the detection of toxic chemicals from human bone marrow is often used in cases with an extended post mortem interval; however, in veterinary medicine, this practice is not used. Therefore, this study was performed to investigate the suitability of bone marrow for toxicological analysis in dogs and cats. Six animals with suspected poisoning were selected; the carcasses were sent for necropsy, and the organs were collected and preserved in buffered formalin and processed routinely for histological examination. In addition, bone marrow samples from the femur, humerus, and tibia were collected for toxicological analysis by liquid chromatography coupled to mass spectrometry detection (LC-MS). This analysis confirmed the presence of aldicarb, aldicarb sulfone, asulam, carbendazim, chlorpyrifos, dichlorvos, thifensulfuron methyl and trifloxysulfuron-sodium and associated with clinical symptoms and anatomo-histopathological alterations it was recognized the poisonings. It is expected that this study will promote the toxicological investigation of bone marrow and open avenues for the use of this tissue as an option for the detection of toxic chemicals in cases of forensic pathology. RESUMO: Na toxicologia forense, a detecção de substâncias químicas tóxicas provenientes da medula óssea humana é frequentemente usada em casos com intervalo post mortem prolongado; no entanto, na medicina veterinária, essa prática não é utilizada. Portanto, este estudo foi realizado para investigar a utilização da medula óssea nas análises toxicológicas em cães e gatos. Seis animais com suspeita de intoxicação foram selecionados; as carcaças foram enviadas para necropsia e os órgãos foram coletados e preservados em formalina tamponada e processados rotineiramente para exame histológico. Amostras de medula óssea de fêmur, úmero e tíbia foram coletadas para análise toxicológica por cromatografia líquida acoplada à espectrometria de massa-massa (LC-MS). A análise por LC-MS confirmou a presença dos agrotóxicos aldicarbe, aldicarbe sulfona, asulam, carbendazim, clorpirifós, diclorvós, tifensulfuron metil e trifloxisulfuron-sódico, e em associação com sinais clínicos e achados anatomo-histopatológicos comprovou-se as intoxicações. Espera-se que este estudo promova a utilização da medula óssea como uma opção na investigação toxicológica para a detecção de produtos químicos tóxicos em casos de patologia forense.

The Solanum glaucophyllum Desf. has been used to treat and prevent diseases in human and veterinary medicine. On the other hand, plant poisoning causes several bone diseases, among them osteoporosis, which is characterized by osteoblastic hypoplasia. Because the osteoblast is a cell derived from the differentiation of mesenchymal stem cells (MSCs) from bone marrow, the hypothesis is that the plant reduces the osteogenic differentiation of MSCs. The objective of this study was to evaluate the effects of S. glaucophyllum Desf. extract on MSCs cultured in osteogenic differentiation medium. We determined by liquid chromatography that 1 ml of plant extract contained 3.8 μl of 1,25(OH)2 D3 (calcitriol). Four groups of MSCs cultivated in osteogenic medium were evaluated as follows: (a) treated with 100 μl of extract/L containing 0.4 μg/L of calcitriol; (b) treated with 1 ml of extract/L containing 4 μg/L of calcitriol; (c) treated with 5 ml of extract/L containing 20 μg/L of calcitriol; and (d) a control group without extract. We performed alkaline phosphatase activity assay, analysis of MTT conversion to formazan, and evaluated the percentage of cells, and number and diameter of mineralization nodules. The expression of gene transcripts for osteopontin, bone sialoprotein and BMP-2 was analysed by RT-qPCR. After 21 days, there was a significant reduction in MTT conversion to formazan in treated groups, of the cellularity in the group with 5 ml of extract/L, and in the number and size of mineralization nodules in the groups treated with 1 and 5 ml of extract/L. The 5 ml extract/L concentration also reduced transcript expression of osteopontin. It is concluded that S. glaucophyllum Desf. at concentrations of 1 and 5 ml extract/L reduced mineralized matrix synthesis in MSCs cultivated in osteogenic differentiation medium, which suggests that this is one of the mechanisms by which osteoporosis occurs in intoxicated animals.

Triple-negative tumors are characterized immunohistochemically by the absence of positivity to sex hormone receptors and to human epidermal growth factor receptor 2. Additionally, they are differentiated into basal-like and non-basal (or null) subtypes, based on the presence of basal cytokeratin expression (CK5/6, 14, and17). Triple-negative subtypes are yet to be characterized in male dogs, to our knowledge. We report herein the clinical and pathologic findings and molecular characterization of carcinoma in the mammary glands of 2 male dogs. Case 1 was diagnosed as a grade II tubulopapillary carcinoma; case 2 was diagnosed as a grade II carcinoma in a mixed tumor. The tumors were characterized phenotypically as triple-negative basal and triple-negative non-basal, respectively.

Objective We sought to evaluate the effect of different concentrations of ethanol on phenotype and activity of articular chondrocyte synthesis of neonatal rats in 2-dimensional (2D) and 3-dimensional (3D) culture. Methods Chondrocytes were cultured in chondrogenic medium with different concentrations of ethanol: 0.0% v/v (control); 0.05% v/v (8.6 mM); 0.25% v/v (42.9 mM), and 0.5% v/v (85.7 mM). Chondrocytes under 2D culture were subjected to MTT assay, while chondrocytes under 3D culture were processed for paraffin inclusion and stained by periodic acid Schiff (PAS) to evaluate mean chondrocyte diameter and percentages of cells, nucleus, cytoplasm, well-differentiated matrix, and PAS+ areas. The expression of gene transcripts for aggrecan, Sox9, and type II collagen was evaluated by real-time quantitative polymerase chain reaction. Results There was no difference between groups by the MTT assay. PAS staining revealed that chondrocytes treated with 0.5% v/v ethanol had higher percentages of cytoplasm and nuclear areas, but with a reduction in PAS+ matrix area. The mean diameter of chondrocytes was similar between groups. The expression of aggrecan in the group treated with 0.5% v/v ethanol was lower in comparison to that in the control. In the groups treated with 0.25% v/v and 0.5% v/v ethanol, the percentage of differentiated cartilage was lower in comparison with that in the control. The group treated with 0.05% v/v ethanol was similar to the control in all parameters. Conclusions Ethanol acted directly on in vitro cultured articular chondrocytes of newborn rats, altering the chondrocyte phenotype and its synthesis activity, and these effects were dose dependent.

The Kisspeptin/Kiss1r system is a key regulator of reproduction by stimulating gonadotrophinreleasing hormone and luteinizing hormone release, and in vitro studies have shown that Kisspeptin can modulate angiogenesis and immune function, factors that are also essential for reproduction However, there are no studies on the expression of Kisspeptin/Kiss1r at the maternal-fetal interface in domestic cats and its relationship with angiogenic and immunological mediators. Thus, our objective was to evaluate the spatiotemporal expression profile of Kisspeptin/Kiss1r and angiogenic and immunological mediators in the uterus and placenta of domestic cats during pregnancy. Uterus and placenta samples were collected from cats in mid pregnancy (N = 6) and late pregnancy (N = 6), in addition to uterus from non-pregnant cats in diestrus (N = 7), to evaluate protein and gene expression of kisspeptin (Kiss1), kisspeptin receptor (Kiss1r), vascular endothelial growth factor (VEGF), tyrosine kinase receptor (Flk-1), placental growth factor (PLGF), interferon gamma (INFγ), migration inhibiting factor (MIF), tumor necrosis factor (TNFα), interleukins (IL6 and IL10) by immunohistochemistry and quantitative polymerase chain reaction. Pregnancy increased the uterine expression of Kiss1 and Kiss1r, especially at the late pregnancy, in addition to upregulating INFy, MIF, Vegf, Il10, and Tnf and downregulating Plgf. Higher placental expression of Kiss1r and Plgf mRNA occurred at the late pregnancy, while the expression of Kiss1, VEGF, Flk-1, INFy, TNFα, Il6, and IL10 was higher in the mid of pregnancy. A positive correlation between Kiss1 and Tnf was observed in the placenta, while Kiss1r had a negative correlation with Infγ, Il6, and Il10. The findings reveal that Kisspeptin/Kiss1r and angiogenic and immunological mediators at the maternal-fetal interface of pregnant cat have a gene correlation and are modulated by the gestational age. These data suggest possible functional links of Kisspeptin in placental angiogenesis and immunology.Summary sentencePregnancy in cats stimulates the uterine expression of the Kiss1/Kiss1r system, especially in late pregnancy, and Kiss1/Kiss1r and angiogenic and immune factors at the cat's placenta have a gene correlation and are modulated by the gestational age.

Failures in hypothalamic kisspeptin/Kiss1r signaling are associated with infertility, and in vitro studies have shown that kisspeptin can modulate angiogenesis and immune activity. Because there is no in vivo research on the functional relationship between these factors in the reproductive system, especially in domestic cats, we evaluated the expression profile of kisspeptin/Kiss1r and angiogenic and immunological mediators in the genital tract of cyclic cats and of those with pyometra. The uterus of cats in diestrus exhibited greater gene and protein expression of Kiss1, as well as Vegf, Pigf, Mif, and Il6. In contrast, Kiss1r presented greater expression in proestrus/estrus, similarly to that observed for the immunostaining of INFγ, MIF, TNFα, and IL10. These factors were positively correlated with Kiss1 and/or Kiss1r, and a positive correlation between Kiss1 and Kiss1r was also observed in the uterus of cats during the estrous cycle. Cats with pyometra showed greater immunostaining of Kiss1 and Kiss1r on the endometrial surface and reduced immunostaining of Kiss1 in deep glands, whereas there was a significant reduction in Vegf, Pigf, Mif, and Il6 mRNA, and an increase in Tnf mRNA. The findings reveal that there is a gene correlation between kisspeptin/Kiss1r and angiogenic and immune mediators in the uterus of the domestic cat, which is modulated by the estrous cycle, and that pyometra affects the expression of these mediators. This study suggests, for the first time, a functional relationship between the Kiss/Kiss1r system and angiogenic and immune mediators in the female genital tract.

The use of stem cells in cell therapies has shown promising results in the treatment of several diseases, including diabetes mellitus, in both humans and animals. Mesenchymal stem cells (MSCs) can be isolated from various locations, including bone marrow, adipose tissues, synovia, muscles, dental pulp, umbilical cords, and the placenta. In vitro, by manipulating the composition of the culture medium or transfection, MSCs can differentiate into several cell lineages, including insulin-producing cells (IPCs). Unlike osteogenic, chondrogenic, and adipogenic differentiation, for which the culture medium and time are similar between studies, studies involving the induction of MSC differentiation in IPCs differ greatly. This divergence is usually evident in relation to the differentiation technique used, the composition of the culture medium, the cultivation time, which can vary from a few hours to several months, and the number of steps to complete differentiation. However, although there is no “gold standard” differentiation medium composition, most prominent studies mention the use of nicotinamide, exedin-4, ß-mercaptoethanol, fibroblast growth factor b (FGFb), and glucose in the culture medium to promote the differentiation of MSCs into IPCs. Therefore, the purpose of this review is to investigate the stages of MSC differentiation into IPCs both in vivo and in vitro, as well as address differentiation techniques and molecular actions and mechanisms by which some substances, such as nicotinamide, exedin-4, ß-mercaptoethanol, FGFb, and glucose, participate in the differentiation process.

This study aimed to evaluate the effects of thyroid hormone on the decidua and metrial gland of rats and to examine the expression of angiogenic factors. 72 adult, female rats were divided into hypothyroid, T4-treated2, and control groups. At 10, 14 and 19 days of gestation (DG), the decidua and metrial gland were collected for histomorphometric and immunohistochemical evaluation of the expression of VEGF, Flk-1 and Tie-2. Hypothyroidism reduced the area of the decidua at 10 and 19 DG. Furthermore, VEGF was increased at 10 and 14 DG, and Flk-1 only at 14 DG, but both was reduced at 19 DG in the metrial gland without significantly changing the area occupied by blood vessels. Rats treated with T4 showed an increase in the decidua blood vessels at 10 and 19 DG. However, at 10 DG, excess T4 resulted in increased of Flk-1 in the decidua and metrial gland. Hypothyroidism increased the Tie-2 at 10 and 19 DG in the decidua and metrial gland. In conclusion, hypothyroidism reduces the area of the decidua and increases the expression of VEGF, Tie-2 and Flk-1. The excess of T4 promotes tissue angiogenesis by increasing the number of vessels in the decidua because of the increased expression of Flk-1.

The hypothesis of this experiment is that mesenchymal stem cells (MSCs) are involved in the genesis of the bone metaplasia caused by Solanum glaucophyllum intoxication. We determined using liquid chromatography that 1 mL of plant extract contained 3.8 μl of 1,25(OH)2D3. The ability of 100 μL, 1 mL and 5 mL of extract/L, containing 1 nM (0.4 μg/L), 10 nM (4 μg/L) and 50 nM (20 μg/L) of 1,25(OH)2D3, respectively, in inducing the osteogenic differentiation in bone marrow MSCs from rats was tested. At the concentrations of 1 and 5 mL of extract/L of culture medium without osteogenesis-inducing factors, the plant extract induced the osteogenic differentiation of the MSCs, as was evidenced by the greater synthesis of mineralized matrix. At the higher concentration (5 mL of extract/L), an increase in the relative expression of BMP-2 gene was observed. It was concluded that rat bone marrow MSC culture is a good model for studying the effects of the S. glaucophyllum extract on the osteogenic differentiation of undifferentiated cells. Also, S. glaucophyllum extracts containing 10 nM (4 μg/L) and 50 nM (20 μg/L) of 1,25(OH)2D3 induce the osteogenic differentiation of MSCs, suggesting that this is one of the mechanisms by which S. glaucophyllum causes bone metaplasia.

Caffeine is an alkaloid that is widely consumed due to its presence in drugs, coffee, tea, and chocolate. This compound passes to offspring through the placenta and milk; can cause teratogenic mutations; and reduces the formation, growth, and mass of bone. Because mesenchymal stem cells (MSCs) are responsible for generating the entire skeleton, we hypothesized that these cells are targets of caffeine. This study evaluated the osteogenic differentiation of MSCs derived from the offspring of rats treated with caffeine during pregnancy and lactation. Twenty-four adult Wistar rats were randomly divided into four groups, including one control group and three experimental groups treated with 25, 50, or 100 mg/kg of caffeine. At weaning, three 21-day-old pups from each dam in each group were euthanized for extraction of bone marrow cells for in vitro tests. Caffeine doses of 50 and 100 mg/kg significantly reduced the activity of alkaline phosphatase at 7, 14, and 21 days and the expression of collagen I at 21 days. However, the expression of gene transcripts for alkaline phosphatase, Runx-2, and bone sialoprotein, as well as the synthesis of mineralization nodules, decreased significantly in all groups treated with caffeine. The expression of osteocalcin was significantly reduced only in the group treated with 50 mg/kg caffeine. The caffeine that passes from the mother to the offspring during pregnancy and lactation reduces the osteogenic differentiation of MSCs. We propose that this reduction in the osteogenic potential of MSCs may be involved in the pathogenesis of osteopenia resulting from caffeine consumption.

Osteoporosis is a metabolic disease that affects bone tissue and is highly associated with bone fractures. Typical osteoporosis fracture treatments, such as bisphosphonates and hormone replacement, present important challenges because of their low bioavailability on the site of action. Options to overcome this issue are systems for the local release of therapeutic agents such as bioactive glasses containing therapeutic molecules and ions. These agents are released during the dissolution process, combining the drugs and ion therapeutic effects for osteoporosis treatment. Among the therapeutic agents that can be applied for bone repair are strontium (Sr) ion and phytopharmaceutical icariin, which have shown potential to promote healthy bone marrow stem cells osteogenic differentiation, increase bone formation and prevent bone loss. Submicron Sr-containing bioactive glass mesoporous spheres with sustained ion release capacity were obtained. Icariin was successfully incorporated into the particles, and the glass composition influenced the icariin incorporation efficiency and release rates. In this work, for the first time, Sr and icariin were incorporated into bioactive glass submicron mesoporous spheres and the in vitro effects of the therapeutic agents release were evaluated on the reduced osteogenic potential of rat osteoporotic bone marrow mesenchymal stem cells, and results showed an improvement on the reduced differentiation potential.

The effects of two concentrations of triiodothyronine (T3; 0.01 and 1,000 nM) on the osteogenic and chondrogenic differentiation abilities of equine adipose-derived mesenchymal stem cells (AD-MSCs) were evaluated. The osteogenic study evaluated the effect of T3 using alkaline phosphatase activity (ALP) assay; cell viability and density; and formation of mineralized nodules at Days 7, 14, and 21 in culture. The chondrogenic study tested the effect of T3 through ALP assay, mitochondrial metabolism, cell density, and periodic acid-Schiff-positive (PAS+) matrix percentage at Days 7 and 14. In both experiments, analysis of variance was used to compare averages through the Student-Newman-Keuls test. In the osteogenic study, no differences in any variable were detected between groups at Day 7. At Day 14, 0.01 nM T3 reduced cell density and the number of mineralized nodules despite the increase in ALP activity and mitochondrial metabolism (P.05). ALP activity increased at 1,000 nM T3 concentration (P.05). At Day 21, 0.01 nM T3 treatment increased ALP activity compared with control treatment (P.05). At 1,000 nM concentration, T3 reduced mitochondrial metabolism and cell density (P.05). In the chondrogenic study, the two T3 concentrations increased cell density compared with control treatment at Day 7. At Day 14, higher T3 concentration reduced mitochondrial metabolism, ALP activity, cell density, and PAS+ chondrogenic matrix percentage compared with control treatment (P.05). Thus, T3 addition to equine AD-MSC cultures has no enhancement effect on osteogenic or chondrogenic differentiation and may, in fact, negatively affect cell density and matrix synthesis depending on hormone concentration and culture time.

O presente relato descreve os achados patológicos relacionados à uma rara apresentação macroscópica de efusão pleural linfocítica maligna em um gato. Macroscopicamente, observou-se efusão pleural branco-amarelada repleta de coágulos brancacentos de células neoplásicas e uma massa tumoral macia e esbranquiçada no mediastino, linfonodos mediastinais e saco pericárdico. As características citológicas do líquido pleural demonstraram um grande número de linfócitos malignos que caracterizam a efusão pleural linfocítica maligna. Histologicamente, a massa tumoral era composta de células redondas com citoplasma eosinofílico e homogêneo variando de escasso a moderado. Os achados macro, micro e citológicos confirmam o diagnóstico de efusão pleural linfocítica maligna devido ao linfoma.

EnglishThe aim of this study was to evaluate the use of mesenchymal stem cells (MSC) in the treatment of myonecrosis induced by Bothrops alternatus venom in rats. Seventy-five male adult Wistar rats were divided into three experimental groups. G1 and G2 were injected in the gastrocnemius muscle with 120μg of B. alternatus venom, while G3 received 200μL of PBS only. Three days after the venom injection, 12 rats from G1 were treated with 5.0 x 106 MSC in PBS, whereas G2 and G3 rats received PBS. Every three days, blood and muscle samples of five animals from each group were taken for serum biochemical and pathological analyses. Histological examinations showed more intense muscle lesions following MSC treatment, characterized by disorganization and loss of muscle fibers, with focal necrosis and inflammatory infiltration by mononuclear cells. In conclusion, the use of MSC for the treatment of local damage caused by inoculation of B. alternatus venom impaired muscle regeneration and interfered in the healing process. Key words: Viperidae envenomation; stem cells; rats; myonecrosis; muscular repair portuguesO objetivo do presente trabalho foi avaliar a utilizacao das celulas tronco mesenquimais (MSC) no tratamento da mionecrose induzida pelo veneno de Bothrops alternatus em ratos. 75 ratos Wistar adultos foram distribuidos em tres grupos experimentais. G1 e G2 receberam 120μg de veneno de B. alternatus, enquanto o G3 recebeu apenas 200μL de PBS. Tres dias apos a administracao do veneno, os ratos do grupo G1 foram tratados com 5.0 x 106 MSC, enquanto G2 e G3 receberam exclusivamente PBS. A cada tres dias, amostras de sangue e tecido muscular foram coletadas de cinco animais de cada grupo para avaliacao bioquimica serica e patologica, respectivamente. A analise histologica revelou lesao muscular mais intensa apos a aplicacao das MSC, caracterizada pela desorganizacao e perdas das fibras musculares, com necrose focal e infiltrado inflamatorio mononuclear. E possivel concluir que a utilizacao das MSC para o tratamento da lesao local causada pela inoculacao do veneno B. alternatus deteriorou a regeneracao muscular e interferiu com o processo de cicatrizacao. Palavras-chave: envenenamento por cobra; celulas tronco; ratos; mionecrose; reparacao muscular

Objective Evaluate the effects of doses of caffeine administered to pregnant rats on the articular cartilage chondrocytes of their offspring. Methods Twenty-four adult Wistar rats were randomly assigned to four groups, with one control group and three groups being treated with caffeine at doses of 25, 50 and 100 mg/kg throughout pregnancy. At birth, three offspring/females were euthanized so that the chondrocytes could be extracted. At 7, 14 and 21 days of culture, the chondrocytes were subjected to the MTT cell viability assay and an evaluation of their alkaline phosphatase activity and collagen synthesis. Chondrocytes were also stained by Hematoxylin-eosin, PAS, Safranin-O and Alcian Blue. The Sox-9, Runx-2, aggrecan, collagen-II and alkaline phosphatase gene transcript levels were also evaluated. Mean comparisons were performed by the Student–Newman–Keuls test. Results Chondrocyte cultures from the 25 mg/kg group had the lowest results, as chondrocytes from this group had reduced viability, percentage of cells, alkaline phosphatase activity and collagen and chondrogenic matrix synthesis. A reduced expression of Sox-9, alkaline phosphatase and collagen-II was also detected in the 25 mg/kg group. Chondrocyte cultures of the group treated with 50 mg/kg caffeine showed reduced collagen synthesis and Sox-9 expression. The caffeine dose of 100 mg/kg also reduced collagen and Sox-9 and alkaline phosphatase expression. Conclusion Caffeine administered to pregnant rats negatively alters the articular cartilage chondrocytes of their offspring, reducing the synthesis of collagen and Sox-9 expression regardless of the dose. This study also concluded that the effects of caffeine are not linear or dose-dependent.

New Findings What is the central question of this study? Clinical studies suggest that obesity ‘protects’ against osteoporosis. However, these studies used only bone densitometry and assessed only one bone site, which is insufficient to enable conclusions to be drawn about the response of the whole skeleton. Furthermore, the effects of exercise on bone responses in obesity have not been explored previously. What is the main finding and what is its importance? We show that obesity causes osteopetrosis. Therefore, the classical perspective of ‘protective effects of obesity’ needs to be reviewed, and exercise is an important tool to avoid these alterations and to maintain the homeostasis of bone. A sedentary lifestyle and obesity induce systemic inflammatory responses. Although the effects of physical inactivity on osseous tissue have been well established, the effects of obesity on bone tissue remain controversial. Furthermore, the effects of physical training on bone tissue responses in the presence of diet-induced obesity are unknown. Our aim was to investigate the effects of obesity and physical training at multiple bone sites in rats. Female Wistar rats were divided into the following four groups: (i) control diet, non-trained (C-NT); (ii) high-refined carbohydrate-containing diet, non-trained (HC-NT); (iii) control diet, trained (C-T); and (iv) high-refined carbohydrate-containing diet, trained (HC-T). At 5 months of age, the rats were submitted to daily exercise for 30 min day−1. After 13 weeks, blood samples, adipose and skeletal tissues were harvested. Two-way ANOVA was applied to detect differences (significance accepted when P ≤ 0.05). The HC-NT group exhibited increased body mass, adiposity, serum leptin, serum insulin, insulin resistance index and concentrations of tumour necrosis factor-α and interleukin-6. Obese rats (HC-NT) exhibited thickening of nasal bones, trabecular bones in the lumbar vertebrae and long bones in a site-dependent manner. The HC-T group exhibited similar adiposity and inflammatory results. Morphological analysis of the lumbar vertebrae in rats fed the HC diet revealed characteristics of osteopetrosis that were inhibited by exercise. In conclusion, the HC diet induced obesity and inflammatory/hormonal alterations and increased the trabecular bone in a site-dependent manner. However, obesity caused osteopetrosis in the lumbar vertebrae, which could be inhibited by physical training. Although exercise inhibited the development of bone alterations, physical training did not inhibit the HC diet-induced obesity responses.

The objective of this study was to establish a protocol for the isolation and culture of mesenchymal stem cells (MSC) from bone marrow and adipose tissue of dogs. Three 6-month-old male dogs were used. Approximately 3.0 cm3 of adipose tissue and 3.0 mL of bone marrow were collected. The samples were processed and the isolated cells were cultured in DMEM. The cells were subjected to phenotypic characterization and to osteogenic, adipogenic, and condrogenic differentiation to confirm the isolation of the MSC. The cells showed elongated and fusiform morphology and they were able to differentiate into osteoblasts, adipocytes, and chondrocytes. Phenotypic characterization revealed high expression of the MSC markers CD90(80.04%) and CD29(96%) in the cells from bone marrow and high expression of CD90(60.94%) and CD29(77.08%) in the cells from adipose tissue. In addition, phenotypic characterization revealed low expression of hematopoietic markers CD45(1.45%) and CD34(1.53%) in the cells from bone marrow and low expression of CD45(1.45%) and CD34(1.53%) in the cells from adipose tissue. Based on these results, the modifications applied to classical protocols simplified the process and proved to be efficient in the isolation, culture, and expansion of MSC isolated from the bone marrow and adipose tissue of dogs. Keywords: cell culture; differentiation; regenerative medicine.

ObjectivesVerify the in-vitro effect of triiodothyronine (T3) on the chondrogenic differentiation of female rat bone marrow mesenchymal stem cells (BMMSCs) over several time periods and at several doses.MethodsCD54 + /CD73 + /CD90 + BMMSCs from Wistar female rats were cultured in chondrogenic medium with or without T3 (0.01; 1; 100; 1000 nm). At seven, 14 and 21 days, the cell morphology, chondrogenic matrix formation and expression of Sox9 and collagen II were evaluated.Key findingsThe dose of 100 nm did not alter the parameters evaluated in any of the periods studied. However, the 0.01 nm T3 dose improved the chondrogenic potential by increasing the chondrogenic matrix formation and expression of Sox9 and collagen II in at least one of the evaluated periods; the 1 nm T3 dose also improved the chondrogenic potential by increasing the chondrogenic matrix formation and the expression of collagen II in at least one of the evaluated periods. The 1000 nm T3 dose improved the chondrogenic potential by increasing the chondrogenic matrix formation and Sox9 expression in at least one of the evaluated periods.ConclusionsT3 has a dose-dependent effect on the differentiation of BMMSCs from female rats.

Purpose/Aim: The aim of this study was to evaluate the effects of excess maternal and postnatal thyroxine on chondrocytes and the extracellular matrix (ECM) of growth cartilage.We used 16 adult female Wistar rats divided into two groups: thyroxine treatment and control. From weaning to 40 days of age, offspring of the treated group (n = 8) received L-thyroxine. Plasma free T4 was measured. Histomorphometric analysis was performed on thyroids and femurs of all offspring. Alcian blue histochemical staining and real-time reverse transcription polymerase chain reaction measurements of gene expression levels of Sox9, Runx2, Aggrecan, Col I, Col II, Alkaline phosphatase, Mmp2, Mmp9, and Bmp2 were performed. Data were analyzed for statistical significance by student's t-test.Excess maternal and postnatal thyroxine reduced the intensity of Alcian blue staining, altered the number of chondrocytes in proliferative and hypertrophic zones in growth cartilage, and reduced the gene expression of Sox9, Mmp2, Mmp9, Col II, and Bmp2 in the growth cartilage of all offspring. Additionally, excess thyroxine altered the gene expression of Runx2, Aggrecan and Col I, and this effect was dependent on age.Excess thyroxine in neonates suppresses chondrocyte proliferation, stimulates chondrocyte hypertrophy and changes the ECM composition by reducing the amount of proteoglycans and glycosaminoglycans (GAGs). Prolonged exposure to excess thyroxine suppresses chondrocyte activity in general, with a severe reduction in the proteoglycan content of cartilage and the expression of gene transcripts essential for endochondral growth and characteristics of the chondrocyte phenotype.

Objective The aim of this study was to unravel the mechanisms by which thyroxine affects skeletal growth by evaluating proliferative activity and angiogenic profile of growth cartilage of neonatal and weanling rats. Methods Sixteen adult Wistar rats were equally divided into 2 groups: control and treated with thyroxine during pregnancy and lactation. The weight, measurement of plasma free T4 and thyroids, femurs' histomorphometric analysis, and proliferative activity and angiogenic profile by immunohistochemical or real-time reverse transcriptase-polymerase chain reaction in growth cartilage was performed. Data were analyzed using Student's t test. Results The free T4 was significantly higher in the treated rats. However, the height of the follicular epithelium of the thyroid in newborns was significantly lower in the treated group. The excess maternal thyroxine significantly reduced the body weight and length of the femur in the offspring but significantly increased the thickness of trabecular bone and changed the height of the zones of the growth plate. Furthermore, excess maternal thyroxine reduced cell proliferation and vascular endothelial growth factor (VEGF) expression in the growth cartilage of newborn and 20-day-old rats ( P < 0.05). There was also a reduction in the immunohistochemical expression of Tie2 in the cartilaginous epiphysis of the newborns and FLK-1 in the articular cartilage of 20-day-old rats. No significant difference was observed in Ang2 expression. Conclusions The excess maternal thyroxine during pregnancy and lactation reduced endochondral bone growth in the progeny and reduced the proliferation rate and VEGF, Flk-1, and Tie2 expression in the cartilage of growing rats without altering the mRNA expression of Ang1 and Ang2.

Resumo: O objetivo deste estudo foi comparar o potencial osteogênico das células tronco mesenquimais extraídas da medula óssea (CTM-MO) com as do tecido adiposo (CTM-AD) de cães adultos. As células foram caracterizadas fenotipicamente quanto à expressão de CD29, CD90, CD34 e CD45 e submetidas à diferenciação adipogênica e condrogênica por 21 dias e osteogênica por 7, 14 e 21 dias. Foram constituídos quatro grupos: 1) CTM-MO em meio osteogênico, 2) CTM-MO em meio basal, 3) CTM-AD em meio osteogênico e 4) CTM-AD em meio basal. Aos 7, 14 e 21 dias de diferenciação osteogênica as culturas foram submetidas às avaliações da conversão de MTT em formazan, da atividade da fosfatase alcalina (FA), da síntese de colágeno e de matriz mineralizada, avaliação do número de células por campo e foram quantificados os transcritos gênicos para osterix, sialoproteina óssea (BSP), osteonectina (ON) e osteocalcina (OC). Tanto as células extraídas da medula óssea quanto do tecido adiposo mostraram elevada expressão de marcadores para células tronco e baixa expressão de marcadores de células hematopoiéticas (menor que 2%). Além disso, foram capazes de se diferenciar em osteoblastos, condrócitos e adipócitos. As CTM-AD submetidas à diferenciação osteogênica mostraram maior conversão do MTT em formazan que as CTM-MO, sob mesmas condições aos 7 e 21 dias. O número de células por campo, a atividade da FA, a síntese de colágeno e de matriz mineralizada foram superior nas CTM-AD em diferenciação, em relação às CTM-MO sob as mesmas condições, em todos os tempos estudados. As expressões de osterix, BSP e OC foram predominantemente superiores nas CTM-MO diferenciadas, mas a expressão de ON foi superior nas CTM-AD diferenciadas aos 7, 14 e 21 dias. Conclui-se que as CTM-AD apresentam maior potencial osteogênico que as CTM-MO quando extraídas de cães adultos. Abstract: The aim of this study was to compare the osteogenic potential of mesenchymal stem cells obtained from bone marrow (BM-MSC) with those extracted from adipose tissue (AT-MSC) of adult dogs. The cells were phenotypically categorized according to the expression of CD29, CD90, CD34 and CD45, and submitted to adipogenic and chondrogenic differentiation for 21 days and osteogenic differentiation for 7, 14 and 21 days. Four groups were formed: BM-MSC in osteogenic medium (1), BM-MSC in basal medium (2), AT-MSC in osteogenic medium (3) and ATMSC in basal medium (4). On days 7, 14 and 21 of osteogenic differentiation, the cultures were submitted to evaluations of MTT conversion in formazan, of alkaline phosphatase activity (AP), of collagen and mineralized matrix synthesis, evaluation of the number of cells per field and there was quantification of the gene transcripts for osterix, bone sialoprotein (BSP), osteonectin (ON) and osteocalcin (OC). Both the cells obtained from bone marrow and those from adipose tissue showed high expression of stem cells markers and low expression of hematopoietic cells markers (lower than 2%). Besides, they were able to differentiate into osteoblasts, chondrocytes and adipocytes. AT-MSC submitted to osteogenic differentiation showed higher MTT conversion in formazan than BM-MSC, under the same conditions on days 7 and 21. The number of cells per field, the AP activity, the collagen and mineralized matrix synthesis were higher in AT-MSC en differentiation, in relation to BM-MSC under the same conditions in all evaluated times. Expressions of osterix, BSP and OC were predominantly higher in differentiated BMMSC, however the expression of ON was higher AT-MSC differentiated on days 7, 14 and 21. In conclusion, AT-MSC present higher osteogenic potential than BM-MSC when extracted from adult dogs.

Objective: To evaluate, in rat offspring, bone changes induced by excess maternal thyroxin during pregnancy and lactation, and to assess the reversibility of these changes after weaning. Material and methods: Twenty Wistar rats were distributed in two groups, hyperthyroid and control, that were treated daily with L-thyroxin (50 mcg/animal) and placebo, respectively. The treatment was initiated seven days before mating and continued throughout pregnancy and lactation. From every female of each of the two groups, two offspring were euthanized after birth, two at 21 days of age (weaning), and two at 42 days of age (21 days after weaning). In newborns, the length of pelvic and thoracic limbs were measured, and in the other animals, the length and width of the femur and humerus were measured. Bones were dissected, decalcified, embedded in paraffin, and analyzed histomorphometrically. Results: Excess maternal thyroxin significantly reduced the length of the pelvic limb in neonates. In 21-day-old individuals, excess maternal thyroxine reduced the length and the width of the femur and the humerus. It also increased thickness of the epiphyseal plate and the percentage of trabecular bone tissue. In 42-day-old individuals, there were no significant differences between groups in relation to the parameters evaluated in the previous periods. Conclusion: Excess maternal thyroxine reduced growth in suckling rats both at birth and at weaning, and it also increased the percentage of trabecular bone tissue in 21-day-old animals. These changes, however, were reversible at 42 days, i.e., 21 days after weaning. Objetivo: Avaliar, na prole de ratos, as alterações ósseas induzidas pelo excesso de tiroxina materna durante gravidez e lactação, e avaliar a reversibilidade dessas alterações após o desmame. Materiais e métodos: Vinte ratos Wistar foram distribuídos em dois grupos, hipertireoide e controle, que foram tratados diariamente com L-tiroxina (50 mcg/animal) e placebo, respectivamente. O tratamento foi iniciado sete dias antes do acasalamento e continuou durante toda a gravidez e lactação. De cada fêmea de cada um dos dois grupos, dois filhotes foram sacrificados após o nascimento, dois aos 21 dias de idade (desmame), e duas aos 42 dias de idade (21 dias após o desmame). Em recém-nascidos, o comprimento dos músculos pélvico e torácico membros foram medidos, e nos demais animais, o comprimento e a largura do fêmur e do úmero foram medidos. Os ossos foram dissecados, descalcificados, incluídos em parafina e analisados ​​histomorfometricamente. Resultados: O excesso de tiroxina materna reduziu significativamente o comprimento do membro pélvico em neonatos. Em indivíduos de 21 dias de idade, o excesso de tiroxina materna reduziu o comprimento e a largura do fêmur e do úmero. Também aumentou a espessura da placa epifisária e a porcentagem do tecido ósseo trabecular. Em indivíduos com 42 dias de idade, não houve diferenças significativas entre grupos em relação aos parâmetros avaliados nos períodos anteriores. Conclusão: Excesso materno A tiroxina reduziu o crescimento em ratas lactantes tanto ao nascimento quanto ao desmame, e também aumentou a porcentagem de tecido ósseo trabecular em animais com 21 dias de idade. Essas mudanças, no entanto, foram reversíveis em 42 dias, ou seja, 21 dias após o desmame.

O trabalho descreve os achados anatomo-histopatológicos e radiológicos do esqueleto de um gato com nanismo associado à adenomatose folicular tireoidiana. Uma gata de 40 dias com pequena estatura e pesando 100g foi encaminhada para a necropsia com histórico de crescimento ósseo severamente reduzido e suspeita de nanismo. Na necropsia, os ossos de todo o esqueleto eram curtos e frágeis. No RX, os ossos longos apresentaram epífises radioluscentes. Microscopicamente, foram observadas características de osteoporose e interrupção do crescimento endocondral, intramembranoso e aposicional, caracterizando o nanismo proporcional. A tireoide apresentava folículos predominantemente pequenos revestidos por células cuboidais e com uma pequena quantidade de colóide. Entre os folículos, havia grande quantidade de células foliculares sem formação de lúmen como característica da adenomatose interfolicular. Com base nos achados macro e microscópicos, o diagnóstico de nanismo proporcional associado à adenomatose folicular tireoidiana foi confirmado.

Background Endometriosis is ectopic development of endometrial glands and stroma in extra-uterine sites and if the lesions occur as a well-defined mass is referred to as endometrioma. In the literature, endometrioma has been reported in only women and there are no reports of endometrioma in animals, including non-human primates. Case presentation A rare case of endometrioma is reported in an 11-year-old female German Shepherd with clinical signs of dehydration, anemia and prostration. Necropsy revealed ascites, generalized pallor, and a well-demarcated reddish mass adjacent to the left ovary and uterus and adherent to the retroperitoneum. The mass measured 25.0 × 20.0 cm with intermingled soft and firm areas. Upon incision, the mass was found to be solid with variable sized cystic cavities filled with coagulated blood. Microscopically, the mass was composed of cuboidal or prismatic epithelial cells arranged in tubules or acini. The epithelium of the mass had similar characteristics to the normal endometrium with PAS-positive secretions. The stroma was prominent and formed by loose connective tissue and smooth muscle fibers as confirmed by Masson trichrome. Extensive multifocal areas of hemorrhage were also observed in the stroma of the mass and in the interior of some epithelium-lined, cystic structures. Most of the epithelial cells had strong and diffuse cytokeratin expression, and some had vimentin expression. Epithelial and stromal cells also showed ERβ, AR, VEGF and COX2 expression. The stroma showed areas with strong and diffuse vimentin expression. Factor VIII expression was observed only in the endothelium of blood vessels in the stroma. Conclusions The macroscopic, microscopic and immunohistochemical findings are consistent with an endometrioma.

Morphological and immunohistochemical characterization of angiogenic and apoptotic factors and the expression of thyroid receptors in the ovary of tilapia Oreochromis niloticus in captivity were studied. The morphological evaluation of the ovaries was performed by histological paraffin embedded and stained with HE. The immunohistochemical expressions of CDC47, VEGF, Flk-1, angiopoietin, Tie-2 and thyroid receptor (TRα) were performed by the technique of streptavidein-biotin-peroxidase. Apoptosis was assessed using the TUNEL kit. The relative expression of thyroid hormone receptors (TRα and TRβ) was assessed by RT-PCR real time. The nuclear expression of CDC47 increased with the stage of maturation of the oocyte and was observed in the follicle cells. Apoptotic bodies were observed in the follicular cells of atretic follicles and postovulatory follicles from the ovaries of 150g and 350g fish. Expression of VEGF and its receptor Flk-1 was also observed in the follicular cells, and the expression of both increased with the maturity of the oocyte, with a higher intensity observed in the full-grown follicle. The expression of angiopoietin and of its receptor (Tie 2) was discrete and moderate respectively. TRα expression was independent of follicular development. However, the 350 g tilapia exhibited higher expression of TRβ compared with the 50 g tilapia. We conclude that the proliferative activity and the expression of VEGF and its receptor increase with follicular maturation and that the TRs expression increases with ovarian maturity in tilapia (Oreochromis niloticus). Foram estudadas as caracterizações morfológica e imuno-histoquímica de fatores angiogênicos e apoptóticos e a expressão de receptores tireoidianos no ovário de tilápia Oreochromis niloticus de cativeiro. A avaliação morfológica dos ovários foi realizada por cortes histológicos incluídos em parafina e corados por HE. As expressões imuno-histoquímicas de CDC47, VEGF e seu receptor Flk-1, angiopoetina e seu receptor Tie-2 e recertor tireoidiano (TRα) foram realizadas pela técnica de estreptavideina-biotina-peroxidade. A apoptose foi avaliada utilizando-se kit de TUNEL. A expressão relativa dos receptores de hormônios tireoidianos (TRα e TRβ) foi avaliada pela técnica de RT-PCR tempo real. A expressão nuclear de CDC47 aumentou com a fase de maturação do oócito e foi observada nas células foliculares. Corpos apoptóticos foram observados nas células foliculares de folículos atrésicos e folículos pós-ovulatórios de ovários de peixes com 150g e 350g. A expressão de VEGF e do seu receptor Flk-1 foi também observada nas células foliculares , e a expressão de ambos aumentou com a maturidade do oócito , com uma maior intensidade no folículo maduro. A expressão de angiopoietina e do seu receptor (Tie 2) foi discreta e moderada, respectivamente. A expressão de TRα foi independente do desenvolvimento folicular. No entanto, a tilápia de 350g apresentou maior expressão de TRβ em comparação com a tilápia de 50g. Conclui-se que a atividade proliferativa e a expressão de VEGF e de seu receptor aumenta com a maturação folicular e que a expressão dos TRs aumenta com a maturidade do ovário em tilápia (Oreochromis niloticus).

Background Caffeine is an active alkaloid that can cause damage to bones in formation during prenatal life into adulthood. This compound can pass across the placenta and into the mother’s milk, causing a reduction in bone formation, growth and mass. The objective of this study was to examine the osteogenic potential of osteoblasts extracted from neonatal rats born to mothers treated with caffeine throughout pregnancy. Methods Twenty-four adult Wistar rats were randomly divided into four groups, consisting of one control group and three groups that were treated with 25, 50, or 100 mg/kg of caffeine by an oral-gastric probe throughout the duration of the experimental period (pregnancy). At birth, three puppies from each dam in each group were euthanized, and osteoblasts were extracted from the calvaria of these pups for in vitro testing. Results The osteoblasts extracted from the pups of rats that received 50 mg/kg caffeine during pregnancy exhibited increased expression of osteocalcin, osteopontin, sialoprotein, runx-2, alkaline phosphatase and type I collagen transcripts, resulting in increased synthesis of mineralization nodules. Conclusions Neonates from rats treated with 50 mg/kg caffeine during pregnancy contained osteoblasts with a higher osteogenic potential characterized by increased expression of osteocalcin, osteopontin, sialoprotein, runx-2, alkaline phosphatase and type I collagen and increased synthesis of mineralization nodules.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A tireoide, por meio da ação dos hormonios tireoidianos, é essencial para a formação e o crescimento do esqueleto. O principal hormônio secretado pela tireoide é a tiroxina (T4), que é convertida em triiodotironina (T3) pela atividade das enzimas iodotironina desiodase D1 e D2 (Bassett e Williams, 2003). Os hormônios tireoidianos (HTs) atuam diretamente no osso, por meio de receptores nucleares específicos (TRα e TRβ), que modulam o desenvolvimento e o metabolismo desse órgão (Wojcicka et al., 2013). Dessa forma, alterações nos níveis séricos dos HT, como acontecem no hipo e no hipertireoidismo, podem resultar em um série de alterações na formação e no crescimento ósseo (Waung et al., 2012; Wojcicka et al., 2013; Bassett e Williams, 2018). As disfuncões tireoidianas podem ocorrer em qualquer fase da vida, mas são comuns durante a gestação. O hipertireoidismo materno tem incidência estimada de 0,2%, podendo chegar até 2,5% quando são considerados os casos de hipertireoidismo materno subclínico (Taylor et al., 2018). O hipertireoidismo materno pode levar ao desenvolvimento fetal anormal, uma vez que ocorre passagem transplacentária de T4, e os HT têm ação crítica em todos os sistemas, particularmente sob a formação e o crescimento ósseo (Brent, 2000; Bassett e Williams, 2016; Gouveia et al., 2018). Durante o desenvolvimento esquelético, a formação óssea ocorre através de dois mecanismos independentes e distintos. Os ossos do crânio, da pelve e o terço lateral do clavícula, formam-se por ossificação intramembranosa, na qual células tronco mesenquimais se diferenciam em osteoblastos. Por outro lado, a maioria dos ossos, inclusive os ossos longos, se formam por meio da formação óssea endocondral, na qual há o desenvolvimento de um molde cartilaginoso, a partir do processo de condrogênese, que será posteriormente substituído por osso (Williams e Bassett, 2018). Os precursores mesenquimais se diferenciam em condrócitos, que proliferam e secretam uma matriz contendo colágeno tipo II (COL2) e proteoglicanos, formando o molde de cartilagem. Os condrócitos se proliferam, diferenciam em condrócitos hipertróficos e sofrem apoptose. Posteriormente, ocorre invasão vascular e migração de osteoblastos, formando o centro de ossificação primário. Mais tardiamente, formam-se os centros de ossificação secundária nas extremidades dos ossos longos, que permanecem separados do centro de ossificação primária pela placa epifisária, onde a ossificação endocondral continua até a vida adulta (Bassett e Williams, 2018; Williams e Bassett, 2018). O crescimento ou ossificação intramembranosa, por sua vez, tem início com a condensação 19 das células tronco mesenquimais que progressivamente se diferenciam em osteoblastos, responsáveis por secretar a matriz osteoide, rica em colágeno tipo I (COL1), que posteriormente mineraliza dentro dos centros de ossificação (Bassett e Williams, 2016; Williams e Bassett, 2018). Bebês expostos ao hipertireoidismo materno apresentam menor comprimento ao nascimento quando comparados à bebês nascidos de mães eutireoideas (Ohrling et al., 2014). Da mesma forma, camundongos com fenótipo de hipertireoidismo apresentam comprimento ósseo encurtado (O’Shea et al., 2003). Estudos prévios demonstraram que o excesso de tiroxina materna reduz o crescimento ósseo da prole por causar várias alterações na cartilagem articular e na placa epifisária dos ossos longos, dentre elas, a de inibir a proliferação de condrócitos, estimular a hipertrofia dos condrócitos da placa epifisária e alterar a composição da matriz extracelular (Ribeiro et al., 2018a). Contudo, o hipertireoidismo materno não só afeta a ossificação endocondral, como também a ossificação intramembranosa. A circunferencia do crânio de bebês expostos ao hipertireoidismo materno é significativamente menor do que em bebês de mães eutireoideas (Ohrling et al., 2014). Essa redução está relacionada ao fechamento precoce das suturas cranianas (Rasmussen et al., 2007; Carmichael et al., 2015). Howie et al., (2016) demonstraram que filhotes de camundongos expostos ao hipertireoidismo materno apresentam alteração na morfologia cranial (Ohrling et al., 2014), com expressão elevada de marcadores da osteogênese, como o Runx2 e a fosfatase alcalina nas suturas cranianas. O processo de formação e crescimento ósseo envolve uma ação coordenada e complexa de proliferação, diferenciação e atividade de vários tipos de células, incluindo condrócitos e osteoblastos (Maes e Kronenberg, 2012). Avanços na compreensão da biologia celular foram possibilitados pelo uso de vários modelos de cultura in vitro, que permitem uma melhor compreensão da fisiopatologia das células envolvidas (Jackson, Sundquist e Marks, 1996; Czekanska et al., 2012). Embora haja pesquisas que demonstraram os efeitos da adição dos hormônios tireoidianos em culturas de condrócitos (Bohme et al., 1992; Alini et al., 1996; Ishikawa et al., 1998; Miura et al., 2002; Robson et al., 2002) e osteoblastos (Ernst e Froesch, 1987; Ohishi et al., 1994; Cray et al., 2013), esse parece ser o primeiro estudo que investigou as culturas de condrócitos e de osteoblastos extraídos de ratos filhos de mães com hipertireoidismo durante a gestação. Neste contexto, o que se pretende é ampliar os conhecimentos, por meio de estudos com condrócitos e osteoblastos de neonatos submetidos ao hipertireoidismo materno, uma vez que os resultados in vitro limitam-se aos efeitos diretos do hormônio, sem levar em consideração seu efeito em todo o organismo, e principalmente neste caso, em que o excesso do hormônio tireoidiano passa da mãe para o feto, através da placenta.

FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais Foram realizados dois estudos para avaliar os efeitos do consumo materno de etanol sobre os condrócitos da cartilagem articular e na diferenciação osteogênica das células tronco mesenquimais da medula óssea (CTM-MO) da prole. Foram utilizadas 13 ratas Wistar adultas distribuídas em dois grupos experimentais, grupo controle e o grupo tratado com etanol. As ratas do grupo etanol e controle, receberam por gavagem diária, a partir do nono dia de gestação, solução alcoólica 40% e água destilada, respectivamente até o trigésimo dia de lactação. No dia do parto, foram eutanasiados quatro neonatos por fêmea e, aos 30 dias de lactação, três filhotes por fêmea. O primeiro estudo avaliou, in vitro, o efeito do consumo materno de etanol sobre os condrócitos da cartilagem articular. Os condrócitos foram extraídos das cartilagens articulares e cultivados em meio condrogênico por sete, 14 e 21 dias. Foram realizados os testes de conversão do MTT, atividade de fosfatase alcalina, porcentagem de células por campo e porcentagem de áreas PAS+ em cultura 3D. Aos 21 dias, foi realizada a quantificação dos transcritos gênicos para agrecano, Sox-9, colágeno tipo II, colágeno X, Runx-2 e VEGF pelo RT-PCR em tempo real. O segundo estudo avaliou o efeito do consumo materno de etanol durante a gestação e lactação sobre a diferenciação osteogênica CTM-MO dos filhotes ao desmame. As CTM-MO foram extraídas dos membros pélvicos e cultivadas em meio osteogênico por sete, 14 e 21 dias. Foram realizados os testes de conversão do MTT, atividade de fosfatase alcalina e porcentagem de células por campo. Aos 21 dias, foram realizados a quantificação do tamanho médio dos nódulos mineralizados e a quantificação dos transcritos gênicos para osteopontina, osteocalcina, BMP-2, Runx-2 e colágeno tipo I por RT-PCR em tempo real. As médias foram comparadas pelo teste t de student e as diferenças foram consideradas significativas se p

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior As plantas calcinogênicas têm importância na medicina veterinária mundial por conterem glicosídeos, que após a sua ingestão, liberam metabólitos da vitamina D, potencialmente nocivos. No Brasil, as espécies Solanum glaucophyllum (S. malacoxylon) e Nierembergia veitchii são responsáveis pela calcinose enzoótica, intoxicação de caráter endêmico que acomete ruminantes. A calcinose é caracterizada pela calcificação de tecidos moles e está frequentemente associada à metaplasias óssea e cartilaginosa. A hipótese do primeiro experimento é que as células tronco mesenquimais (CTM) estejam envolvidas na gênese do processo de metaplasia óssea causado pela Solanum glaucophyllum, o que ainda carece de comprovação científica. Além da metaplasia, a intoxicação pela Solanum glaucophyllum causa alterações ósseas, representadas por osteopetrose, osteonecrose e osteoporose. Por ser o osteoblasto uma célula que deriva da diferenciação osteogênica das CTM da medula óssea, a hipótese do segundo experimento é que a osteoporose induzida pela intoxicação por Solanum glaucophyllum seja decorrente não somente da ação da planta sobre os osteoblastos, como também do seu efeito sobre a diferenciação osteogênica das CTM. A concentração de 1,25(OH)2D3 obtida, por meio da técnica de cromatografia líquida, no extrato da planta foi de 3,8 µg/ml. Em ambos os experimentos, foram testados os efeitos de 100µl, 1ml e 5ml de extrato/L, contendo respectivamente, 1nM (0,4 µg/L), 10nM (4 µg/L) e 50nM (20 µg/L) de 1,25(OH)2D3. Foram realizados os seguintes ensaios: ensaio de MTT, atividade de fosfatase alcalina, determinação da porcentagem de células/campo, número de nódulos de mineralização em 50 campos e avaliação da expressão dos transcritos gênicos para osteopontina, sialoproteína óssea e BMP-2, por RT-PCR tempo real. No primeiro experimento,culturas de CTM da medula óssea foram utilizadas como modelo para estudo dos efeitos da Solanum glaucophyllum na indução da diferenciação osteogênica de células indiferenciadas. Na concentração de 5ml de extrato de Solanum glaucophyllum por litro de meio de cultura desprovido de fatores indutores da osteogênese, o extrato da planta induziu diferenciação osteogênica das CTM, comprovada pela maior síntese de matriz mineralizada e pelo aumento da expressão relativa dos transcritos gênicos para BMP-2. A diferenciação osteogênica induzida pelo extrato da planta foi estatisticamente semelhante à diferenciação induzida por meio de cultura contendo fatores que comprovadamente induzem osteogênese, representados pelo β-glicerofosfato, ácido ascórbico e pela dexametasona. Conclui-se que culturas de CTM da medula óssea de ratos são um bom modelo para estudar o efeito do extrato da Solanum glaucophyllum sobre a diferenciação osteogênica de células indiferenciadas e que os extratos de Solanum glaucophyllum contendo 10nM (4 µg/L) e 50nM (20 µg/L) de 1,25(OH)2D3 causam diferenciação osteogênica de CTM, sugerindo ser este um dos mecanismos pelo qual a Solanum glaucophyllum produz metaplasia óssea. No segundo experimento, a fim de estudar os efeitos da Solanum glaucophyllum em CTM submetidas à diferenciação em osteoblastos, diferentes concentrações do extrato da planta foram adicionadas ao meio de cultura, juntamente com fatores conhecidamente promotores de diferenciação osteogênica, representados pelo β-glicerofosfato, ácido ascórbico e pela dexametasona. Aos 21 dias, nas concentrações de 1ml e de 5ml de extrato/L, houve redução significativa do número e do tamanho dos nódulos de mineralização, respectivamente. A concentração de 5ml de extrato/L também reduziu significativamente a expressão do transcrito para osteopontina em comparação ao controle. Conclui-se que o extrato de Solanum glaucophyllum apresenta efeito negativo sobre culturas de células tronco mesenquimais comprometidas com a diferenciação osteogênica, o que sugere ser este um dos mecanismos pelo qual ocorre osteoporose nos animais intoxicados pela planta.

A adição de hormônios, fatores de crescimento, vitaminas, dentre outros em culturas de células tronco mesenquimais (CTM) tem o objetivo de aumentar a proliferação, induzir a diferenciação e diminuir o tempo de cultivo. Além disso, células tronco (CT) com maior potencial osteogênico ou condrogênico são células com maior capacidade de sintetizar, in vitro, matriz mineralizada e cartilaginosa, respectivamente. O efeito da adição da triiodotironina (T3) em culturas de CTM sob diferenciação osteogênica e condrogênica já foi testado com bons resultados em células de ratos e humanos. Assim, uma hipótese é que o uso de CT, com maior potencial de diferenciação osteogênico ou condrogênico, poderia aumentar as chances de recuperação ou acelerar o tratamento de doenças ou alterações que afetam os tecidos ósseo e cartilaginoso, respectivamente. O objetivo deste estudo foi avaliar o efeito da T3 em duas concentrações (0,01nM e 1000nM) comparadas ao grupo controle sobre a diferenciação osteogênica e condrogênica das CTM do tecido adiposo (CTM-TA) de equinos. No primeiro experimento, avaliou-se o efeito da T3 sobre a diferenciação osteogênica das CTM-TA, por meio dos ensaios de avaliação da atividade da fosfatase alcalina (ALP); da viabilidade celular e da densidade celular, bem como a formação de nódulos mineralizados aos sete, 14 e 21 dias de cultivo. No segundo experimento, avaliou-se o efeito da T3 sobre a diferenciação condrogênica das CTM-TA, através da avaliação da atividade da fosfatase alcalina; do metabolismo mitocondrial; da densidade celular e da porcentagem de matriz condrogênica aos sete e 14 dias. Em ambos os experimentos, foi realizada ANOVA com comparação das médias pelo teste SNK. Diferenças entre grupos foram consideradas significativas se p