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This review aims to discuss the main factors involved in the development of early antral follicles until gonadotropin dependence. This follicular phase is characterized by intense proliferation of granulosa cells, formation of a fluid-filled cavity, morphological differentiation of cumulus cells, mural granulosa cells and recruitment of theca cells. The interaction between oocyte, granulosa and theca cells is crucial for follicular growth and hormone production. Growth factors produced by the oocyte, such as growth and differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15), regulate granulosa cell proliferation and differentiation and antral cavity development, as well as stimulate the production of follicle-stimulating hormone (FSH) receptors in granulosa cells. In response to FSH, granulosa cells secrete C-type natriuretic peptide (CNP), which acts through its receptor to increase cyclic guanosine monophosphate (cGMP) production and consequently follicular development. Granulosa cells also produce insulin-like growth factor-1 (IGF-1) and increase aromatase enzyme activity, which results in greater sensitivity to gonadotropins and follicular steroidogenesis. The absence of IGF-1 signaling causes cessation of follicular growth at the early antral stage. Many other local factors are involved in the regulation of follicular development. Therefore, this review brings relevant data for a better understanding of the mechanisms involved in the control of early antral follicle growth, emphasizing the role of endocrine and paracrine factors, the oocyte-granulosa cell interaction and the processes of follicular atresia. The challenges for the establishment of efficient culture systems for in vitro growth of early antral follicles are also discussed.

Esta revisão tem como objetivo discutir os principais fatores envolvidos no desenvolvimento de folículos antrais iniciais até a dependência de gonadotrofinas. Essa fase folicular é caracterizada por intensa proliferação de células da granulosa, formação de uma cavidade preenchida por líquido, diferenciação morfológica das células do cumulus, células da granulosa murais e recrutamento de células da teca. A interação entre oócito, células da granulosa e da teca é determinante para o crescimento folicular e produção hormonal. Fatores de crescimento produzidos pelo oócito, fator de crescimento e diferenciação-9 (GDF-9) e proteína morfogenética óssea-15 (BMP-15), regulam a proliferação e diferenciação de células da granulosa, e o desenvolvimento da cavidade antral, bem como estimulam a produção de receptores do hormônio folículo estimulante (FSH) nas células da granulosa. Em resposta ao FSH, as células da granulosa secretam o peptídeo natriurético tipo C (CNP), que atua através de seu receptor para aumentar a produção de monofosfato de guanosina cíclico (GMPc) e consequentemente o desenvolvimento folicular. As células da granulosa também produzem o fator de crescimento semelhante à insulina 1 (IGF-1) e aumentam a atividade da enzima aromatase, o que resulta em maior sensibilidade às gonadotrofinas e esteroidogênese folicular. A ausência de sinalização do IGF-1 causa cessação do crescimento folicular no início do estágio antral. Muitos outros fatores locais estão envolvidos na regulação do desenvolvimento folicular. Por tanto essa revisão traz dados relevantes para uma melhor compreensão dos mecanismos envolvidos no controle do crescimento de folículos antrais iniciais, enfatizando o papel dos fatores endócrinos e parácrinos, a interação oócito-células da granulosa e os processos de atresia folicular. Os desafios para o estabelecimento de sistemas de cultivo eficientes para o crescimento in vitro de folículos antrais iniciais também são discutidos.

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GnRH analogues were widely used for controlld ovary stimulation, but their effects on oocyte quality remain contradictory. This study aimed to explore the influence of GnRH analogues on oocyte quality in mice. A total of 120 mice were randomly assigned to four groups:(i)GnRH-a+PMSG group; (ii) GnRH-ant+PMSG group; (iii) PMSG group; (iv) Control group. Ovaries were collected for quantitative real-time polymerase chain reaction (qRT-PCR) to assess GDF9 and BMP15 mRNA expression, and protein expression were evaluated by western blotting. Moreover, embryo developmental progress in vitro and implantation rate in vivo were recorded. Compared with control group, both GDF9 mRNA and protein expressions were strengthened in PMSG group, but reduced in the presence of GnRH-a or GnRH-ant. The GnRH-a group exhibited decreased BMP15 mRNA expression compared to PMSG group, while the GnRH-ant group did not show the same pattern. BMP15 protein expression were not statisticlly different among the four groups. Notably, there was no statistically difference in the expression of these two factors between GnRH-a and GnRH-ant groups. The percentage of zygotes progressing to the 2-cell stage and percentage of 2-cell advancing to the blastocyst stage were similar in the PMSG group and control group. However, both the GnRH-a and GnRH-ant groups showed decreased embryos development rates compared to other two groups. The embryonic implantation rate in control group (53.3%) was higher than that in the GnRH-a and GnRH-ant groups (33.3% and 30.8%, P<0.05). The difference between the PMSG (45.0%) and GnRHa group was statistically significant (P value of 0.023), but not between the PMSG and GnRH-ant group (P value of 0.486). No statistical difference was confirmed between GnRH-a and GnRH-ant groups. Our findings shed light on the safety of GnRH analogues in ovary stimulation, and highlight the need for further research to establish optimal and effective controlled ovary stimulation protocol.(AU)

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Litter size is one of the crucial factors in livestock production and is of high economic value, which is affected by ovulation rate, hormones, and growth factors.Growth factors play a multifaceted role in reproductive physiology. This review aims to investigate the association of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9)with litter size in livestock.The transforming growth factor ß(TGF-ß) superfamily includes more than 34 members; GDF9 and BMP15 are among the most significantfactors for regulating fertility and litter size in most livestock species. Ovarian follicles release BMP15 and GDF9 that are involved in the maturation of primary follicles into the basal form, proliferation of granulosa and theca cells, steroidogenesis, ovulation, and formation of the corpus luteum. Besides, these factors are highly expressed in oocytes and are necessary for female fertility and multiple ovulation in several livestock species. Animals with two inactive copies of these factors are sterile, while those with one inactive copy are fertile. Thus, the present reviewprovides valuable information on the association of BMP15 and GDF9with litter size in livestock that can be used as biological markers of multiple ovulation or for improving fertility in livestock.(AU)

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Purpose: To investigate the mechanism of polysaccharides from aloe vera (PAV), a main active ingredient of Aloe vera, treatment in pulpitis rats. Methods: Pulpitis were modeled by drilling the occlusal central fossa with Sprague Dawley rats. Next, the rats were treated with 20, 40, and 80 mg/kg PAV for three weeks, respectively. Computed tomography scanning assay, hematoxylin and eosin staining, and tartrate-resistant acid phosphatase staining were used to detect the pathology change. Then, levels of tumor necrosis factor-α, interleukin-1ß, prostaglandin E2, and ciclooxigenase 2 were detected by enzyme-linked immunosorbent assay. The expressions of bone morphogenetic protein 2 human (BMP-2), osteocalcin, osterix, and runt-related transcription factor 2 (Runx2) were quantified by quantitative real-time polymerase chain reaction and Western blotting (WB). Finally, Wnt3a expression, p-GSK3ß/GSK3ß and p-ß-catenin/ß-catenin ratio were analyzed by WB. Results: PAV up regulated the bone mineral density, and reduced the breakage of the crown and cervical structures, and the necrosis of the crown and root pulp of pulpitis rats. In addition, results indicated that PAV could inhibit osteoblast formation. While osteoblasts' number was decreased, proteins of BMP-2, osteocalcin, osterix, and Runx2 were up-regulated by PAV. Furthermore, PAV increased the Wnt3a expression and the p-ß-catenin/ß-catenin ratio, and decreased p-GSK3ß/GSK3ß ratio. Interestingly, these effects were all in dose dependence. Conclusions: PAV could inhibit pulp inflammation and promote osteoblasts differentiation via suppressing the activation of the Wnt/ß-catenin signaling, enhancing the dental bone density.

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The identification of mutations in the genes encoding bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) associated with phenotypes of sterility or increased ovulation rate in sheep aroused interest in the study of the role of local factors in preantral and antral folliculogenesis in different species. An additive mutation in the BMP15 receptor, BMPR1b, which determines an increase in the ovulatory rate, has been introduced in several sheep breeds to increase the number of lambs born. Although these mutations indicate extremely relevant functions of these factors, the literature data on the regulation of the expression and function of these proteins and their receptors are very controversial, possibly due to differences in experimental models. The present review discusses the published data and preliminary results obtained by our group on the participation of local factors in the selection of the dominant follicle, ovulation, and follicular atresia in cattle, focusing on transforming growth factors beta and their receptors. The study of the expression pattern and the functionality of proteins produced by follicular cells and their receptors will allow increasing the knowledge about this local system, known to be involved in ovarian physiopathology and with the potential to promote contraception or increase the ovulation rate in mammals.(AU)

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Research focused on female gamete vitrification has increased attention to develop a reliable cryopreservation method to preserve immature equine oocytes. Despite the intensive implementation of biotechnological procedures for horse breeding, vitrification of immature equine cumulus-oocyte complexes (COCs) remain to be clearly elucidated. We aimed to determine the relative transcript level of target genes Bone morphogenetic protein 15 (BMP15); Bcl-2-associated X protein (BAX); and Caspase 3 (CASP3) in equine COCs prior to and after vitrification. Ovarian follicles were aspirated from ovaries collected from an abattoir. A total of 240 COCs were collected and distributed into vitrified COCs (VIT, n=120) and nonvitrified (Non-VIT, n=120) groups. Then, COCs were preserved and relative transcript expressions of BMP15, BAX, CASP3 were measured and normalized against GAPDH performed by qRT-PCR. In addition, 38 COCs were evaluated to assess chromatin configuration of germinal vesicl e stage prior and after vitrification by exposure to 10 µg/ml of bisbenzimide. A difference was observed in the COCs' mRNA level of abundance for the BAX gene between the VIT (2.05 ± 0.47) and (0.85 ± 0.08) Non-VIT groups. There was no difference in mRNA relative transcript level of CASP3 and BMP15 in Non-VIT (0.63 ± 0.20 and 1.55 ± 0.73, respectively) compared to VIT (0.64 ± 0.01 and 2.84 ± 2.20, respectively) equine COCs. All COCs where considered at immature stage of development even though COCs in Non-VIT group showed higher condensed chromatin configuration compared to VIT (100% vs 60.7%, respectively). We demonstrate that BMP15 and CASP3 are detected in VIT and Non-VIT immature COCs. In conclusion, BAX is expressed highly in vitrified immature equine COCs and indicates that activation of apoptosis signaling cascades in cells exposed to vitrification.(AU)

Pesquisas sobre a vitrificação de gametas femininos estão sendo realizadas para o desenvolvimento de um método confiável de criopreservação dos complexos cumulus-oócitos (CCOs) na espécie equina. Apesar da implementação intensiva de biotecnologias reprodutivas em equinos, a vitrificação dos CCOs imaturos permanece em estágio experimental em relação à competência celular. O objetivo do estudo foi determinar o nível de transcrição relativo dos genes Proteína morfogenética óssea 15 (BMP15); Proteína X associada a Bcl-2 (BAX); e Caspase 3 (CASP3) em CCOs equinos antes e após a vitrificação. Folículos ovarianos foram aspirados de ovários coletados em matadouro. O total de 240 CCOs foi coletado e distribuído em grupos vitrificados (VIT, n=120) e não vitrificados (N-VIT, n=120). Os CCOs foram preservados e as expressões transcritas relativas de BMP15, BAX, CASP3 foram determinadas pela técnica de qRT-PCR sendo normalizadas em relação ao GAPDH. Além disso, 38 CCOs foram avaliados para determinar a configuração da cromatina no estágio de vesícula germinativa antes e após a vitrificação pela exposição a 10 µg/ml de bisbenzimida. Os resultados mostraram uma diferença no nível de abundância de mRNA dos CCOs para o gene BAX entre os grupos VIT (2,05 ± 0,47) e N-VIT (0,85 ± 0,08). Não houve diferença no nível de transcrição relativa do mRNA de CASP3 e BMP15 nos CCOs do grupo N-VIT (0,63 ± 0,20 e 1,55 ± 0,73, respectivamente) em comparação com VIT (0,64 ± 0,01 e 2,84 ± 2,20, respectivamente). Todos os CCOs foram considerados em estágio imaturo de desenvolvimento, embora os CCOs no grupo N-VIT apresentaram a configuração de cromatina condensada em maior número de células avaliadas em comparação com VIT (100% vs 60,7%, respectivamente). Demonstramos que BMP15 e CASP3 são detectados em CCOs imaturos em VIT e N-VIT. Conclui-se que o BAX é altamente expresso em CCOs equinos imaturos vitrificados sendo relacionado à sinalização de apoptose em células expostas ao processo de vitrificação.(AU)

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Purpose To evaluate the effect of ergosterol combined with risedronate on fracture healing. Methods Sixty male Sprague Dawley fracture model rats were assigned into group A (n=20), group B (n=20), and group C (n=20) at random. All rats were fed by gavage until their sacrifice as it follows: group A with ergosteroside and risedronate, group B with risedronate, and group C with saline solution. At weeks 2 and 4, 10 rats of each group were sacrificed. Healing effect and bone tissue changes in the fractures site were assessed by using hematoxylin and eosin stain histology. Enzyme-linked immunosorbent assay was used to detect the expression of serum bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-7 (BMP-7), and vascular endothelial growth factor (VEGF). Reverse transcriptase polymerase chain reaction was applied to detect the expression of osteoprotegerin (OPG) mRNA, osteocalcin (OCN) mRNA and core-binding factor subunit-?1 (CBF-?1) mRNA. Results In terms of serum BMP-2, BMP-7, and VEGF expression at weeks 2 and 4 after gavage, group A < group B < group C (P<0.05). At week 4 after gavage, serum VEGF expression in the three groups harbored positive relationship with serum BMP-2 and BMP-7 expression (P<0.05). Regarding serum OPG, OCN and CBF-?1 mRNA expression at weeks 2 and 4 after gavage, group A

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Although ovarian aging is a key cause of decreased ovarian function and oocyte quality, it remains a problem in infertility treatment. Therefore, this study is aimed to investigate whether Paeonia lactiflora (PL), a herb improves ovarian function and oocyte quality using aged female mice. C57BL/6 female mice aged 8 months were treated orally every day with PL of 26.5 mg/kg (n=7) and 53 mg/kg (n=7) of body weight for 4 weeks using an oral zoned needle. The control group (n=7) was treated with normal saline. Ovaries and serum were collected for the H&E stain and the evaluation of reactive oxygen species (ROS) levels, respectively. In the second experiment, female mice were orally administered with PL (26.5 mg/kg: n=12, 53 mg/kg: n=12, control: n=12) and then superovulated with PMSG and hCG, and mated with male mice. Zygotes were retrieved and cultured for 4 days. Ovaries were provided for examination of expressions of genes associated with angiogenesis (VEGF and visfatin), anti-aging (Sirt1 and Sirt2), and follicular development (c-Kit, BMP-15, and GDF-9). PL significantly increased numbers of surviving follicles (primordial, primary, secondary, and antral), numbers of zygotes retrieved, embryo development rate, and ovarian expression of VEGF, visfatin, c-Kit, BMP-15, and GDF-9 at both doses. However, ovarian expression of Sirt1 and Sirt2 was increased at 53.0 mg/kg of PL. ROS levels were not affected by PL. These results suggest that PL may possess beneficial effects regarding ovarian function and oocyte quality, possibly by activation of ovarian angiogenesis and follicular development.

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Abstract Purpose: To investigate the protective effects of salvianolic acid A (SAA) on renal damage in rats with chronic renal failure (CRF). Methods: The five-sixth nephrectomy model of CRF was successfully established in group CRF (10 rats) and group CRF+SAA (10 rats). Ten rats were selected as sham-operated group (group S), in which only the capsules of both kidneys were removed. The rats in group CRF+SAA were intragastrically administrated with 10 mg/kg SAA for 8 weeks. The blood urine nitrogen (BUN), urine creatinine (Ucr), creatinine clearance rate (Ccr), and serum uperoxide dismutase (SOD) and malondialdehyde (MDA) were tested. The expressions of transforming growth factor-β1 (TGF-β1), bone morphogenetic protein 7 (BMP-7) and Smad6 protein in renal tissue were determined. Results: After treatment, compared with group CRF, in group CRF+SAA the BUN, Scr, serum MDA and kidney/body weight ratio were decreased, the Ccr and serum SOD were increased, the TGF-β1 protein expression level in renal tissue was decreased, and the BMP-7 and Smad6 protein levels were increased (all P < 0.05). Conclusion: SAA can alleviate the renal damage in CRF rats through anti-oxidant stress, down-regulation of TGF-β1 signaling pathway and up-regulation of BMP-7/Smad6 signaling pathway.

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Background: The Green iguana (Iguana iguana) is a reptile belonging to the Iguanidae family. It is an ectothermic animalwith arboreal habits and a daytime activity pattern. Leaves, fruits, and eggs are part of their diet. These animals can be foundin the South, North and Central America. Free-living Green iguanas may suffer stress during environmental changes, whichcan lead to a homeostatic imbalance. There is a correlation between stress and anorexia which results in an increase in theoccurrence of fractures. Reptile fractures are generally treated by providing rigid stabilization and alignment maintenance.The present study reports the use of locking-plate osteosynthesis in one iguana.Case: One female green iguana, weighing 1.690 kg, was assisted at the Hospital Veterinário (Hovet) - Federal da Universidade Federal de Mato Grosso (UFMT). During anamnesis, it was observed that this iguana was a non-captive animal,which had fallen from a tree. The animal was unable to perform physical movements with the forearm displaying bonecrepitation. It was also observed apathy and dehydration. The iguana was subjected to a range of supplementary examinations and on the x-ray image, it was detected that there was a complete right humerus fracture. Following examination,the animal underwent surgery for fracture stabilization. Humerus osteosynthesis was performed with compression in a 1.5mm 6-hole locking-plate. During the osteosynthesis procedure a morphogenetic graft was inserted. Immediate post-surgeryradiographic evaluation was performed, and that confirmed fracture reduction and bone alignment. The animal displayedclinical improvement after the second post-operative day once it returned to regular ingestion of diet. On the 30th postoperative day, the radiographic evaluation showed evidence of bone consolidation. On the 40th post-operative day, theanimal displayed satisfactory gait and voluntary ingestion of food, thus enabling its return to...

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The transforming growth factors beta (TGFβ) are local factors produced by ovarian cells which, after binding to their receptors, regulate follicular deviation and ovulation. However, their regulation and function during corpus luteum (CL) regression has been poorly investigated. The present study evaluated the mRNA regulation of some TGFβ family ligands and their receptors in the bovine CL during induced luteolysis in vivo. On day 10 of the estrous cycle, cows received an injection of prostaglandin F2α (PGF) and luteal samples were obtained from separate groups of cows (n= 4-5 cows per time-point) at 0, 2, 12, 24 or 48 h after treatment. Since TGF beta family comprises more than 30 ligands, we focused in some candidates genes such as activin receptors (ACVR-1A, -1B, -2A, -2B) AMH, AMHR2, BMPs (BMP-1, -2, -3, -4, -6 and -7), BMP receptors (BMPR-1A, -1B and -2), inhibin subunits (INH-A, -BA, -BB) and betaglycan (TGFBR3). The mRNA levels of BMP4, BMP6 and INHBA were higher at 2 h after PGF administration (P<0.05) in comparison to 0 h. The relative mRNA abundance of BMP1, BMP2, BMP3, BMP4, BMP6, ACVR1B, INHBA and INHBB was upregulated up to 12 h post PGF (P<0.05). On the other hand, TGFBR3 mRNA that codes for a reservoir of ligands that bind to TGF-beta receptors, was lower at 48 h. In conclusion, findings from this study demonstrated that genes encoding several TGFβ family members are expressed in a time-specific manner after PGF administration.

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ABSTRACT: This study aimed to evaluate the economic efficiency of DD treatment on milk yield in lame cows suffering from DD. A total of 33 Holstein dairy cows with DD were included in the study. The milk yields were assessed as (1st); beginning milk production (BMP), (2nd); peak milk production before the diagnosis (PMPBD), (3rd); diagnosis day milk production (DMP), and (4th); post-treatment milk production (TMP). In the first stage of analyses, using the E-views equity test of means program, cows were classified into three groups for diagnosis time of DD according to the day in milk (DIM) (Group 1: 0 ≤ DIM ≤ 50, Group 2: 51 ≤ DIM ≤ 100, and Group 3: 101 ≤ DIM ≤ 150). Analysis of variance (ANOVA F-Test) and the Welch F-Test were conducted to compare the means of TMP to BMP, PMPBD, and DMP. Differences between TMP and DMP were statistically significant in all three groups. In the second stage of analyses, a cost-benefit analysis was conducted to determine the break-even point for each group to cover treatment costs for increasing milk yield. The DIM of DMP was considered as the gained milk yield per cow. Treatment cost and the mean break-even DIM for each group was then calculated. After treatment, the mean optimum break-even day for Groups 1, 2, and 3 was determined as 18.68, 26.43, and 27.14, respectively. Results suggested that treatment of DD may be considered as favorable and useful for a dairy economy.

RESUMO: Esse estudo tem o objetivo de avaliar a eficiência econômica no tratamento da DD na produção de leite em vacas tratadas com claudicao. Foram incluidas no total 33 vacas leiteiras Holstein com DD nesse estudo. A produção de leite foi avaliada (1°); início da produção de leite (BMP), 2°; pico na produção de leite antes do diagnostico (PMPBD), (3°); dia de diagnostico da produção de leite (DMP), e (4°); produção de leite pós-tratamento (TMP). Na primeira etapa das análises, utilizamos o programa E-views Equity Test of Mean, as vacas foram classificadas em três grupos para o tempo de diagnostico de DD Segundo o dia do leite (DMI) (Grupo 1: 0 ≤ DIM ≤ 50, Grupo 2 : 51 ≤ DIM ≤ 100 e Grupo 3: 101 ≤ DIM ≤ 150).A análise de variância (ANOVA F-Test) e o Welch F-Test foram conduzidos para comparar as medias de TMP a BMP, PMPBD e DMP. Diferenças entre TMP e DMP foram estatisticamente significativas nos três grupos. No segundo estágio das análises, uma análise de custo-benefício foi realizada para determinar o ponto de equilíbrio para cada grupo para cobrir os custos de tratamento e aumentar a produção de leite. O DIM de DMP foi considerado como a produção de leite adquirida por vaca. O custo do tratamento e o DIM médio de equilíbrio para cada grupo foram então calculados. Após o tratamento, o dia de equilíbrio ideal médio para os Grupos 1, 2 e 3 foi determinado como 18,68; 26,43 e 27,14, respectivamente. Os resultados sugerem que o tratamento de DD pode ser considerado favorável e útil para uma economia leiteira.

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The objective was to evaluate the in vitro effect of prolactin in osteogenic potential of adipose tissue-derived mesenchymal stem cells (ADSCs) in female rats. ADSCs were cultured in osteogenic medium with and without the addition of prolactin and distributed into three groups: 1) ADSCs (control), 2) ADSCs with addition of 100ng/mL of prolactin and 3) ADSCs with addition of 300ng/mL of prolactin. At 21 days of differentiation, the tests of MTT conversion into formazan crystals, percentage of mineralized nodules and cells per field and quantification of genic transcript for alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I by real-time RT-PCR were made. The addition of prolactin reduced the conversion of MTT in group 3 and increased the percentage of cells per field in the groups 2 and 3, however without significantly increasing the percentage of mineralized nodules and the expression of alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I. In conclusion, the addition of prolactin in concentrations of 100ng/mL and 300ng/mL does not change the osteogenic differentiation to the ADSCs of female rats despite increase in the cellularity of the culture.(AU)

O objetivo do presente trabalho foi avaliar o efeito in vitro da prolactina sobre o potencial osteogênico de células-tronco mesenquimais do tecido adiposo (CTM-TA) em ratas. CTM-TA foram cultivadas em meio osteogênico com e sem adição de prolactina e distribuídas em três grupos: 1) CTM-TA (controle), 2) CM-TA com adição de 100ng/mL de prolactina e 3) CTM-TA com adição de 300ng/mL de prolactina. Aos 21 dias de diferenciação, foram realizados os testes de conversão do MTT em cristais de formazan, porcentagem de nódulos mineralizados e células por campo e quantificação dos transcritos gênicos para fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. A adição de prolactina reduziu a conversão do MTT no grupo 3 e aumentou a porcentagem de células por campo nos grupos 2 e 3, sem alterar significativamente a porcentagem de nódulos mineralizados e a expressão de fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. Conclui-se que a adição de prolactina nas concentrações de 100ng/mL e 300ng/mL não altera a diferenciação osteogênica das CTM-TA de ratas, apesar do aumento de celularidade da cultura.(AU)

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Polymorphisms in the BMP-15 gene related to Galway (FecXG) and Inverdale (FecXI) and in the BMPR-1B gene known as Booroola (FecB) mutations were investigated using the Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) method, on sheep from the breeds Santa Inês (n= 574) and Morada Nova (n=282). DNA was extracted and amplified through PCR with specific primers that introduced a restriction site in association with the mutation. The PCR products were submitted to endonucleases. The experiment found no FecXG and FecXI mutations. Six samples of animals with multiple offspring/birth history presented polymorphism for FecB similar to control samples, but this pattern was not confirmed by nucleotide sequencing. Although the absence of these mutations in the studied breeds, other factors related to prolificacy should be investigated to explain the inherent prolificity mechanisms.(AU)

Polimorfismos Galway (Fec XG ) e Inverdale (Fec XI), relacionados ao gene BMP-15, e Booroola (FecB), localizado no gene BMPR-1B, foram investigados usando-se a técnica de reação em cadeia da polimerase - polimorfismo de comprimento de fragmentos de restrição (PCR-RFLP), em ovelhas Santa Inês (n= 574) e Morada Nova (n=282). O DNA foi extraído e amplificado por PCR com iniciadores específicos, que introduziram um sítio de restrição associado à mutação, em seguida os amplicons foram submetidos à ação de endonucleases. Não foram observadas as mutações Fec XG e Fec XI nas amostras estudadas. Amostras de seis animais com histórico de partos gemelares apresentaram polimorfismo para FecB semelhantes às amostras controle, mas esse padrão não foi confirmado pelo sequenciamento de nucleotídeos. Apesar da ausência dessas mutações nos animais das raças estudadas, outros fatores relacionados à prolificidade devem ser pesquisados para explicar os mecanismos da alta prolificidade desses animais.(AU)

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As características reprodutivas, tais como o número de tetos, uniformidade e tamanho de leitegada, são essenciais para os programas de melhoramento animal devido á importância para a cadeia produtiva, pois influenciam na habilidade materna das porcas e pode afetar o número de leitões desmamados. Assim, objetivou-se a identificação e anotação funcional de genes candidatos associados a características reprodutivas em suínos. A identificação dos genes candidatos foi realizada por meio de uma revisão sistemática entre dois avaliadores independentes que utilizaram o mecanismo de busca na Web of Science. As consultas de pesquisa utilizaram combinações de palavras-chave seguindo os critérios: termos relacionados às características avaliadas (number of teats, teats number, total number born, litter uniformity, uniformity of the litter); tipos de teste de associação (gwas, genome wide association); e espécie (pig). Para a anotação de variantes alvo foi utilizado o banco de dados público do Ensembl contendo as variantes estruturais de suínos. Em seguida, foram classificadas de acordo com sua potencial função e concatenadas com os estudos prévios de GWAS. Os genes obtidos por meio da revisão sistemática de GWAS para as características reprodutivas de suínos, que possuíram variantes na região 5UTR e/ou codificante, foram utilizados par a construção das redes gênicas de processos biológicos e Gene-Fatores de Transcrição. Da revisão sistemática um total de quatorze artigos foram selecionados, e o processo de anotação do gene resultou em um total de 2.077 genes candidatos. Após combinados com a lista de genes associados com às variantes estruturais, restaram 306 genes com suas potenciais variantes estruturais. Três genes para a característica de tamanho de leitegada foram priorizados como os genes mais candidatos (GRID2, GRIK3 e PALB2), com seus processos biológicos enriquecidos (ex: via de sinalização de receptores ionotrópicos de glutamato e crescimento de blastócito). Para a característica de número de tetos, seis genes foram destacados como os genes mais candidatos (GHR, IFT80, FSTL3, SKOR1, SMURF1 e AKT3), e seus respectivos processos biológicos (ex: receptor do hormônio de crescimento, regulação da via de sinalização de BMP - Bone Morphogenetic Proteins e proliferação de vasos sanguíneos). Com as redes Gene-Fatores de Transcrição, identificamos os principais FTs e os genes candidatos mais relevantes para o tamanho de leitegada (PALB2 e GRID2) e para número de tetos (RIN, LTBP2 e COL6A6). Com isso, a partir deste estudo, identificamos os genes mais promissores associados ao tamanho de leitegada e número de tetos, que poderão ser usados nos estudos de genética e melhoramento animal, pois seus mecanismos genéticos regulatórios, estão envolvidos na expressão fenotípica dessas características, influenciando no desempenho reprodutivo e produtividade suinícola. A descoberta destes genes, podem trazer novas considerações para o conhecimento atual da arquitetura genética dessas características, uma vez que, os marcadores associados a estes genes, podem ser atribuídos com pesos mais elevados na seleção genômica e validados em populações específicas.

Reproductive traits, such as the number of teats, uniformity and litter size, are essential for animal breeding programs due to the importance for the production chain, since they influence the maternal ability of sows and can affect the number of weaned piglets. Thus, the objective was to identify and functionally annotate candidate genes associated with reproductive traits in swine. The identification of candidate genes was carried out through a systematic review between two independent evaluators who used the Web of Science as search tool. The search queries used combinations of keywords following the criteria: terms related to the traits evaluated (number of teats, teats number, total number born, litter uniformity, uniformity of the litter); types of association tests (gwas, genome wide association); and species (pig). The public Ensembl database containing the swine structural variants was used for the annotation of target variants. Then, they were classified according to their potential function and concatenated with genes found in previous GWAS studies. The genes obtained through the systematic review of GWAS for the reproductive traits of swine, which had variants in the 5'UTR and/or coding region, were used to perform the genetic networks of biological processes and Gene-Transcription Factors. From the systematic review, a total of fourteen articles were selected, and the gene annotation process resulted in a total of 2077 candidate genes. After combining the list of genes associated with the structural variants, remained 306 genes with potential structural variants. Three genes for litter size trait were prioritized as the most candidate genes (GRID2, GRIK3 and PALB2), with their biological processes enriched (ex: ionotropic glutamate receptor signaling pathway and blastocyst growth).Six genes were highlighted as the most candidate genes for the teat number trait (GHR, IFT80, FSTL3, SKOR1, SMURF1 and AKT3), and their respective biological processes (ex: growth hormone receptor, regulation of the signaling pathway of BMP - Bone Morphogenetic Proteins and blood vessel proliferation). In the Gene-Transcription Factor networks, we identified the main TFs and the most relevant candidate genes for litter size (PALB2 and GRID2) and teat number (RIN, LTBP2 and COL6A6). Thus, from this study, we identified the most promising genes associated with litter size and number of teats, which could be used in genetic studies and animal breeding, since regulatory genetic mechanisms are involved in the phenotypic expression of these traits, influencing on reproductive performance and swine productivity. The discovery of these genes may bring new considerations to the current knowledge of the genetic architecture of these traits, since the markers associated with these genes can be assigned higher weights in genomic selection and validated in specific populations.

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As lesões tendíneas são causas importantes de claudicação e afastamento do esporte em equinos e no homem. A incidência de lesões é variada de acordo com a modalidade e intensidade atlética, compreendendo cerca de 30% das lesões musculoesqueléticas de ambas as espécies. Os tratamentos convencionais são pouco eficazes em reparar com qualidade a matriz extracelular (MEC), o que determina o elevado índice de recidivas. As células-tronco mesenquimais (CTM) ganharam destaque no tratamento das tendinopatias e demonstram resultados favoráveis nas características histológicas e mecânicas teciduais, bem como redução do tempo de reparação tendínea. Estas caracteristicas terapeuticas das CTM são atribuidas à sua habilidade de imunomodulação, característica anti-inflamatória, promotora da reorganização tecidual e capacidade de diferenciação em linhagens mesodermais. Já é conhecida a possibilidade de diferenciação osteogênica, adipogênica e condrogênica in vitro das CTM, no entanto a diferenciação tenogênica ainda não possui uma metodologia padronizada e eficaz. A modalidade de cultivo tridimensional (3D) de CTM em materiais sintéticos ou biológicos é capaz de estimular a diferenciação celular de acordo com a composição do arcabouço, além de oferecer proteção e potencializar o desempenho terapêutico. Objetivo: Determinar qual melhor fonte de CTM equinas dentre: tecido adiposo (CTMad); medula óssea (CTMmo); e tendão obtidos por isolamento (CTMtd ISO) ou explante (CTMtd EXPL), que possui a melhor capacidade de proliferação in vitro, diferenciação tenogênia em cultura 3D em hidrogel de MEC tendinea equina (HgMECTdEq) e cultivo 2D em membrana Transwell® revestida de colágeno I e III (1:1). Objetivamos também avaliar a eficácia de uma nova metodologia de diferenciação tenogênica. Métodos: As quatro diferentes fontes de CTM foram obtidas de um mesmo equino com 10 meses de idade. As amostras foram processadas e as CTM, isoladas e cultivadas. As células foram caracterizadas através de sua morfologia fibroblastóide, aderência ao plástico, expansão clonal, expressão de receptores de superficie CD13-, CD34-, CD44+, CD45-, CD90+, CD105+, CD106-, MHC-II- e diferenciação osteogênica, adipogênica e condrogênica. A capacidade de proliferação in vitro de cada origem foi avaliada pelos testes de Taxa de Proliferação Celular (TPC) e Tempo de Cuplicação da População Celular (TDPC), cultavas por 72 horas em três diferentes concentrações iniciais (1x104, 3x104 e 1x105). Foi avaliada a habilidade de diferenciação tenogênica de cada origem celular através da produção de colágeno pela coloração de Picrosirius Red quando cultivadas em membrana Transwell® revestida com COL1:COL3 e meio de diferenciação tenogênica (50 ng/ml de Proteína Óssea Morfogenética-12 (BMP-12), 50 g/ml de Ácido Ascórbico e 5 g/ml de ITS). O HgMECTdEq foi produzido de acordo com protocolo desenvolvido pelo Laboratório de Terapias Regenerativas da FMVZ Unesp, Botucatu-SP. Posteriormente 2x105 CTM de cada origem foram adicionadas em cultivo 3D imersas em 0,5 mL de hidrogel e cultivadas por 7 dias em meio de cultivo padrão e de diferenciação tenogênica. Foi determinada a concentração de metaloproteinases de matriz 2 e 9 (MMP-2 e MMP-9) pelo ensaio de zimografia e em análise quantitativa por PCR a expressão gênica dos marcadores de tenogênese Scleraxis, Mohawk, Biglicana, Decorin, Tenascina C, Colágeno I e Colágeno III, além do fator osteogênico RunX2 do cultivo 3D em HgMECTdEq e em cultivo 2D em membrana COL1:COL3, com meio padrão e de diferenciação tenogênica. Resultados: Todas as células apresentaram características similares quanto à morfologia fibroblastoide, aderência ao plástico e expansão clonal. Houve baixa expressão dos receptores de superfície CD105 para CTMtd ISO e CD44 para as CTMad e positiva expressão de MHC-II para as CTMtd EXPL. Superior habilidade de proliferação in vitro foi identificada para a CTMad e inferior capacidade de diferenciação adipogênica e condrogênica foi observada para as CTMtd EXPL. A nova metodologia de diferenciação tenogênica foi capaz de produzir a diferenciação tenogênica das quatro origens de CTM, com inferior produção de colágeno para as CTMmo. O meio de diferenciação tenogênico foi capaz de aumentar a concentração de MMP-2 e MMP-9 e também aumentar a expressão dos marcadores tenogênicos Sclerax, Mohawk, Biglicana, Decorin, COL1, COL3, Tenascina C e o fator esteogênico Runx2. Conclusão: A nova metodologia de diferenciação tenogênica proposta pelo estudo foi eficaz em induzir a tenogênese de todas as fontes de CTM avaliadas. De acordo com nossos resultados a melhor fonte em capacidade de proliferação, diferenciação tenogênica in vitro e seguridade para aplicações clínicas são as CTMad. As CTMtd ISO e EXPL, apesar de sua evidente capacidade de tenogênese, apresentam população heterogênia em cultivo e resultados de caracterização que desencorajam sua aplicação clínica, especialmente de forma alogênica.

Tendon injuries are important causes of lameness and withdrawal from sport in horses and men. The incidence of injuries varies according to the sport and athletic intensity, comprising about 30% of musculoskeletal injuries of both species. Conventional treatments are ineffective in repairing the extracellular matrix (ECM) with quality, which contributes to the high rate of recurrence. Mesenchymal stem cells (MSC) gained prominence in the treatment of tendinopathies and showed favorable results in the histological and mechanical tissue characteristics, as well as a reduction in the tendon repair time. These therapeutic characteristics of MSCs are attributed to their ability to immunomodulate, anti-inflammatory, promote tissue reorganization and differentiate into mesodermal lineages. The possibility of osteogenic, adipogenic and chondrogenic differentiation in vitro of MSCs is already known, however tenogenic differentiation still does not have a standardized and effective methodology. The modality of three-dimensional (3D) culture of MSC in synthetic or biological materials is capable of stimulating cell differentiation according to the composition of the framework, in addition to offering protection and enhancing therapeutic performance. Objective: To determine the best source of equine MSCs among adipose tissue (MSCad), bone marrow (MSCmo) and tendon obtained by isolation (MSCtd ISO) or tendon explant (MSCtd EXPL), for in vitro proliferation and tenogenic differentiation under two different conditions: 3D hydrogel cultivation of equine tendon ECM (HgMECTdEq); and 2D cultivation on a type I and III collagen-coated membrane. We also aim to evaluate the effectiveness of a new formulation of a means inducing tenogenic differentiation. Methods: The four different sources of MSC were obtained from the same 10-month-old horse. Samples were processed and MSCs isolated and cultivated. The cells were characterized by their fibroblastoid morphology, plastic adherence, clonal expansion, expression of CD13-, CD34-, CD44+, CD45-, CD90+, CD105+, CD106-, MHC-II- surface receptors and osteogenic, adipogenic and differentiation. chondrogenic. The in vitro proliferation capacity of each source was evaluated by the Cell Proliferation Rate (CPT) and Population Doubling Time (PDT) tests, grown for 72 hours at three different initial concentrations (1x104, 3x104 and 1x105). The tenogenic differentiation ability of each cell origin was evaluated through the production of collagen by Picrosirius Red staining when cultivated on a Transwell® membrane coated with COL1:COL3 and tenogenic differentiation medium (50 ng/ml of Morphogenetic Bone Protein-12 (BMP) -12), 50 g/ml Ascorbic Acid and 5 g/ml ITS). The HgMECTdEq was produced according to a protocol developed by the Laboratory of Regenerative Therapies at FMVZ Unesp, Botucatu-SP. Afterwards, 2x105 CTM of each origin were added in 3D culture immersed in 0.5 mL of hydrogel and cultivated for 7 days in standard culture medium and tenogenic differentiation. The concentration of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) was determined by zymography assay and in quantitative analysis by PCR the gene expression of tenogenesis markers Scleraxis, Mohawk, Biglican, Decorin, Tenascin C, Collagen I and Collagen III, in addition to the osteogenic factor RunX2 from 3D culture in HgMECTdEq and in 2D culture in COL1:COL3 membrane, with standard culture medium and tenogenic differentiation. Results: All cells showed similar characteristics regarding fibroblastoid morphology, plastic adherence and clonal expansion. There was low expression of surface receptors CD105 for CTMtd ISO and CD44 for CTMad and positive expression of MHC-II for CTMtd EXPL. Superior in vitro proliferation ability was identified for MSCad and inferior capacity for adipogenic and chondrogenic differentiation was observed for MSCtd EXPL. The new tenogenic differentiation methodology demonstrated efficacy in the four MSC origins, with lower collagen production for MSCmo. The tenogenic differentiation medium was able to increase the concentration of MMP-2 and MMP-9 and also increase the expression of tenogenic markers Sclerax, Mohawk, Biglican, Decorin, COL1, COL3, Tenascin C and the osteogenic factor Runx2. Conclusion: According to our results, we determined that the best source of proliferation capacity and tenogenic differentiation in vitro are MSCad. MSCtd ISO showed an evident in vitro tenogenesis capacity, being a promising source of CTM. The new tenogenic differentiation methodology proposed by the study was effective in inducing tenogenesis of all MSC sources evaluated.

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This study evaluated the effect of different concentrations of bone morphogenetic protein-4 (BMP-4), as well as the interaction of BMP-4 and follicle stimulating hormone (FSH) on growth, ultrastructural integrity, and expression of mRNA for growth differentiation factor-9 (GDF-9), BMP-15, maternal antigen that the embryo requires (Mater) and nucleoplasmin-2 (Npm-2) in bovine secondary follicles cultured in vitro for 18 days. Follicles cultured in the presence of 50 ng/ml BMP-4 had a progressive increase in their diameters with the increase of culture period from 0 to 6 and 12 days, but no significant differences were observed among treatments. The presence of both FSH and BMP-4 in a culture medium did not stimulate follicle growth when compared to the control medium. After 12 days, the percentage of normal follicles was maintained similar to that of day 0 in the medium supplemented with both FSH and BMP-4, but no significant differences among treatments were observed after 18 days of culture. BMP-4 maintained the ultrastructural integrity of follicles after 18 days of culture, while follicles cultured in medium supplemented with FSH or both BMP-4 and FSH had oocyte with irregular zona pellucida, vesicular bodies, and an abundance of vacuoles. Follicles cultured in the presence of BMP-4 had an increase in the levels of BMP-15 mRNA, when compared to those cultured in medium supplemented with FSH alone. In conclusion, the addition of BMP-4 in culture medium contributes to preserve follicular ultrastructure, but BMP-4 did not interact positively with FSH. Regarding secondary follicles cultured in the presence of FSH, BMP-4 increases the expression of mRNA for BMP-15.

Resumo

O objetivo do estudo foi avaliar o efeito do consumo materno de etanol durante a gestação e lactação sobre a massa óssea e sobre a diferenciação osteogênica in vitro das células tronco mesenquimais da medula óssea (CTM-MO) de ratas. Foram utilizadas 13 ratas Wistar adultas distribuídas em dois grupos: 1) controle e 2) tratado com etanol. As ratas do grupo etanol e controle, receberam diariamente, a partir do nono dia de gestação, solução alcoólica 40% (4g de etanol/kg) e água destilada, respectivamente até o trigésimo dia de lactação. As CTM-MO foram extraídas dos fêmures e tíbias direitas e cultivadas em meio osteogênico por sete, 14 e 21 dias. Foram realizados os testes de conversão do MTT em cristais de formazan, atividade de fosfatase alcalina e avaliação da porcentagem de células por campo. Aos 21 dias, foram realizadas as quantificações dos nódulos mineralizados por campo e dos transcritos gênicos para osteopontina, osteocalcina e BMP-2 por RT-PCR tempo real. As avaliações morfológicas e morfométricas da porcentagem de osso trabecular e espessura do osso cortical foram realizadas nos fêmures e tíbias esquerdas. As médias foram comparadas pelo teste t de student e as diferenças foram consideradas significativas se p<0,05. As CTM-MO das ratas que consumiram etanol durante a gestação e lactação, quando submetidas a diferenciação osteogênica, in vitro, apresentaram maior conversão de MTT em formazan, maior atividade de fosfatase alcalina, maior porcentagem de células por campo, maior expressão de BMP-2 e maior síntese de nódulos mineralizados quando comparadas às células das ratas controle. Entretanto, não houve diferença significativa entre grupos, na porcentagem de tecido ósseo trabecular e na espessura da cortical. Conclui-se que, o consumo de etanol durante a gestação e lactação não altera os tecidos ósseos trabecular e cortical do fêmur e tíbia em comparação ao de ratas gestantes e lactantes controles, apesar das CTM-MO apresentarem, in vitro, maior diferenciação osteogênica caracterizada pela maior síntese de matriz mineralizada.

The aim of this study was to evaluate the effect of maternal ethanol consumption during gestation and lactation on bone mass and the osteogenic differentiation of mesenchymal stem cells of the bone marrow (BMMSCs) of rats. Thirteen adult Wistar rats were used. The rats were distributed in two groups: 1) control and 2) ethanol treated. The rats of the ethanol and control groups received daily, from the ninth day of gestation, 40% alcoholic solution and distilled water, respectively, until the thirtieth day of lactation. The BMMSCs were extracted from the right femurs and tibiae and cultured on osteogenic medium for 7, 14 and 21 days. The conversion of MTT to formazan crystals, alkaline phosphatase activity, and porcentage of cells per field were performed. The number of mineralized nodules per field and the quantification of the gene transcripts for osteopontin, osteocalcin and BMP-2 by real-time RT-PCR were performed at day 21. Morphometric evaluations of the percentage of trabecular bone and cortical thickness were performed in the left femur and tibia. The means were compared by the Students 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 submitted to osteogenic differentiation in vitro, presented higher conversion of MTT to formazan, higher alkaline phosphatase activity, higher percentage of cells per field, higher expression of BMP-2 and higher synthesis of mineralized nodules when compared to that of control rat cells. However, there was no significant difference in the percentage of trabecular bone or cortical thickness between both groups. It is concluded that 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 in vitro BMMSCs showing greater osteogenic differentiation characterized by greater synthesis of mineralized matrix.

Resumo

Este estudo teve como objetivo avaliar o efeito do concentrado autólogo de plaquetas (CAP) na osteoartrose (OA) induzida experimentalmente em coelhos, através da avaliação histológica, imuno-histoquímica e artroscópica, e simultaneamente, determinar as concentrações plasmáticas do fator de crescimento transformador 1 (TGF-1) e do fator de crescimento derivado das plaquetas (PDGF). Foram utilizados 30 coelhos machos, adultos jovens, saudáveis, da raça Nova Zelândia, com massa corporal média de 3,0kg, submetidos à ruptura do ligamento cruzado cranial (LCCr) (M1) por vídeo artroscopia para a indução da OA. Aos 21 dias depois da ruptura, os coelhos foram submetidos à reconstituição do ligamento (M2) por vídeo artroscopia e foram divididos em dois grupos de acordo com o tratamento: O grupo controle, que recebeu 0,5 ml de solução Ringer Lactato por via intra-articular e o grupo CAP, que recebeu 0,5 ml de concentrado autólogo de plaquetas por via intra-articular. As injeções intra-articulares dos tratamentos foram realizadas imediatamente após a reconstituição do ligamento, aos 15 e aos 30 dias após a reconstituição. Cinco coelhos de cada grupo foram eutanasiados aos 15 dias após a reconstituição (M3), outros cinco aos 30 (M4), e os restantes cinco coelhos de cada grupo aos 60 dias (M5) após a reconstituição. Nesses mesmos momentos, foram feitas avaliações artroscópicas para avaliar a evolução do processo degenerativo e a resposta aos tratamentos aplicados e antes de cada procedimento, foram coletados dois ml de sangue da artéria auricular central e depositados em tubos contendo EDTA e posteriormente acondicionadas em eppendorfes estéreis em duas alíquotas iguais para fazer a dosagem de TGF-1 e PDGF respectivamente. Nos M3, M4 e M5, depois da eutanásia dos animais, foram coletados fragmentos da cápsula sinovial e da cartilagem articular para a avaliação histológica e imuno-histoquímica. Foi observado na avaliação histológica que os animais que receberam o CAP apresentaram, significativamente, (p<0,05) menor inflamação sinovial, menor proliferação celular e menor degradação da matriz extra celular (MEC) quando comparados com os coelhos que receberam Ringer Lactato. Na avaliação imuno-histoquímica os animais que receberam o CAP apresentaram, significativamente, menor expressão (p<0,05) de interleucina 1 (IL-1), de fator de necrose tumoral (TNF), da proteína morfogenética 2 (BMP-2) e, significativamente, maior expressão de PDGF na membrana sinovial quando comparados com os coelhos que receberam Ringer Lactato, e na cartilagem dos animais desse mesmo grupo, expressão, significativamente (p<0,05) menor, de IL-1 e de TNF e maior expressão de BMP-2 e PDGF quando comparados com os animais do grupo controle. Na avaliação artroscópica foi observado nos animais que receberam o CAP melhora significativa (p<0,05) na inflamação da membrana sinovial, menor formação de cordões fibrosos e menos fibrilação da cartilagem. Finalmente, na avaliação da concentração dos fatores de crescimento, não foram observadas diferenças estatisticamente significativas (p>0,05) nas concentrações de TGF-1 em nenhum dos momentos avaliados após as aplicações do CAP, e foi observado diferença estatisticamente significativa na concentração plasmática do PDGF no M5 nos animais do grupo CAP quando comparado com os animais do grupo controle. Segundo os resultados deste experimento, conclui-se que o CAP é capaz de controlar o processo inflamatório que acontece durante a OA melhorando a sinovite presente na cápsula sinovial ao tempo que diminui a degradação da cartilagem articular. Adicionalmente, esse composto biológico regula a síntese e secreção de citocinas inflamatórias e aumenta a liberação do PDGF.

This study aimed to evaluate the effect of autologous platelet concentrate (APC) on osteoarthritis (OA) experimentally induced in rabbits, through histological, immunohistochemical and arthroscopic evaluation, and simultaneously determine plasma concentrations of the transforming growth factor 1 (TGF -1) and platelet-derived growth factor (PDGF). The study used 30 male rabbits, young, healthy, New Zealand breed, with an average body mass of 3.0 kg, and submitted to rupture of the cranial cruciate ligament (CrCL) (M1) by video arthroscopy to induce OA. At 21 days after the rupture, the rabbits were submitted to ligament replacement (M2) by video arthroscopy and were divided into two groups according to the treatment: The control group, which received 0.5 ml of the Ringer Lactate solution by intra-articular injections and the CAP group, which received 0.5 ml of autologous platelet concentrate by intra-articular injections. The intra-articular injections of the treatments were performed immediately after the ligament replacement, at 15 and 30 days after the replacement. Five rabbits from each group were euthanized at 15 days after replacement (M3), another five at 30 (M4), and the remaining five rabbits from each group at 60 days (M5) after replacement. At those same moments, arthroscopic evaluations were carried out to assess the evolution of the degenerative process and the response to the treatments applied and before each procedure, two ml of blood from the central auricular artery were collected and deposited in tubes containing EDTA and later stored in sterile eppendorfs in two equal aliquots to measure TGF-1 and PDGF respectively. In M3, M4 and M5, after euthanasia of the animals, fragments of the synovial capsule and articular cartilage were collected for histological and immunohistochemical evaluation. It was observed in the histological evaluation that the animals that received the CAP had significantly (p <0.05) less synovial inflammation, less cell proliferation and less degradation of the extracellular matrix (ECM) when compared to the rabbits that received Ringer Lactate. In the immunohistochemical evaluation, the animals that received the CAP showed lower secretion (p <0.05) of interleukin 1 (IL-1), tumor necrosis factor (TNF), morphogenetic protein 2 (BMP-2) and greater secretion of PDGF in the synovial membrane when compared to the rabbits that received Ringer Lactate, and in the cartilage of the animals of that same group, significantly lower (p <0.05) IL-1 and TNF secretion and greater BMP-2 synthesis and secretion and PDGF when compared to animals in the control group. In the arthroscopic evaluation, a significant improvement (p <0.05) in inflammation of the synovial membrane, less formation of fibrous cords and less fibrillation of the cartilage was observed in the animals that received CAP. Finally, when assessing the concentration of growth factors, there were no statistically significant differences (p> 0.05) in TGF-1 concentrations in any of the moments evaluated after CAP applications, and a statistically significant difference in plasma concentration was observed of PDGF in M5 in CAP group animals when compared to control group animals. According to the results of this experiment, it is concluded that the CAP is able to control the inflammatory process that occurs during OA, improving the synovitis present in the synovial capsule while decreasing the degradation of the articular cartilage. Additionally, this biological compound regulates the synthesis and secretion of inflammatory cytokines and increases the release of PDGF.

Resumo

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<0,05. Os neonatos das mães que receberam etanol foram menos pesados quando comparados aos neonatos controle. A cultura dos condrócitos do grupo etanol apresentou maior conversão de MTT, maior atividade de fosfatase alcalina e maior porcentagem de células relação ao grupo controle em todos os períodos. Não houve diferença entre ambos os grupos na porcentagem de áreas PAS+ em cultura 3D. Houve maior expressão de colágeno tipo II e menor expressão de Sox-9 no grupo etanol 15 em relação ao grupo controle. O peso dos filhotes, ao desmame, das mães que receberam etanol foi significativamente menor quando comparado ao controle. As CTM-MO do grupo etanol apresentaram menor conversão de MTT aos sete dias de diferenciação osteogênica porém maior atividade de fosfatase alcalina, maior porcentagem de células, maior tamanho médio dos nódulos mineralizados bem como maior expressão de BMP-2, osteopontina e osteocalcina. Conclui-se que o consumo materno de etanol durante a gestação e lactação altera, in vitro, o fenótipo e a atividade de síntese dos condrócitos dos neonatos bem como aumenta a diferenciação osteogênica das CTM-MO da prole ao desmame

Two studies were performed to evaluate the effects of maternal ethanol consumption on articular cartilage chondrocytes and on osteogenic differentiation of offspring bone marrow mesenchymal stem cells (BMMSCs). Thirteen adult female Wistar rats were divided into two experimental groups, the control group and the ethanol-treated group. The rats of the ethanol and control group received by daily gavage, from the ninth day of gestation, 40% alcohol solution and distilled water, respectively until the thirtieth day of lactation. On the day of delivery, four newborns were euthanized per female and, at 30 days of lactation, three pups per female. The first study evaluated in vitro the effect of maternal ethanol consumption on articular cartilage chondrocytes. Chondrocytes were extracted from articular cartilage and cultivated in chondrogenic medium for seven, 14 and 21 days. MTT conversion tests, alkaline phosphatase activity, percentage of cells per field and percentage of PAS+ areas in 3D culture were performed. At 21 days, the quantification of gene transcripts for agrecan, Sox-9, collagen type II, collagen X, Runx-2 and VEGF was performed by real-time RT-PCR. The second study evaluated the effect of maternal ethanol consumption during pregnancy and lactation on BMMSCs osteogenic differentiation of pups at weaning. The BMMSCs were extracted from the pelvic limbs and cultured in osteogenic medium for seven, 14 and 21 days. MTT conversion, alkaline phosphatase activity and cell percentage per field tests were performed. At 21 days, the average size of the mineralized nodules was quantified and the gene transcripts were quantified for osteopontin, osteocalcin, BMP-2, Runx-2 and collagen I by real-time RT-PCR. Means were compared by Student's t-test and differences were considered significant if p <0.05. Neonates of mothers receiving ethanol were less heavy when compared to control neonates. The chondrocyte culture of the ethanol group showed higher MTT conversion, higher alkaline phosphatase activity and higher percentage of cells compared to the control group in all periods. There was no difference between both groups in the percentage of PAS+ areas in 3D culture. There was higher expression of collagen type II and lower expression of Sox-9 in the ethanol group compared to the control group. The weight of pups at weaning of mothers receiving ethanol was significantly lower when compared to control. BMMSCs ethanol group showed lower MTT conversion after seven days of osteogenic differentiation but 17 higher alkaline phosphatase activity, increased percentage of cells larger average size of mineralized nodules as well as increased expression of BMP-2, osteopontin and osteocalcin. In conclusion, maternal ethanol consumption during pregnancy and lactation alters, in vitro, the phenotype and chondrocyte synthesis activity of neonates, as well as increases the osteogenic differentiation of BMMSCs from weaning offspring