Resumo
The aims of this study were to investigate the effects of different concentrations of Platelet-derived growth factor-BB (PDGF-BB) on the survival, activation, levels of ROS, and growth of goat preantral follicles enclosed in ovarian tissue. For this, ovarian fragments were cultured for 7 days in Alpha Minimum Essential Medium (α-MEM+ ) with or without PDGF-BB (0, 25, 50 and 100 ng/ml). The results showed that both the 25 ng/ml PDGF and the 50 ng/ml PDGF treatments maintained the percentage of morphologically normal follicles from day 1 to day 7. In addition, the 25 ng/ml PDGF treatment showed a significantly higher percentage of morphologically normal follicles when compared to the other treatments. At day 7, greater (P < 0.05) follicular and oocyte diameters were observed in the 25 ng/ml PDGF and the 50 ng/ml PDGF treatments when compared to the cultured control treatment. On day 7 of culture, all the treatments tested had a significant increase in the percentage of developing follicles when compared to the non-cultured control. However, the percentage of follicle activation, as well as ROS production, were similar (P < 0.05) among the treatments, irrespective of culture time. In conclusion, PDGF-BB improved, in a concentration-dependent manner, follicular survival as well as oocyte and follicular diameter after in vitro culture of goat preantral follicle-enclosed in ovarian tissue fragments.
Assuntos
Animais , Cabras/anatomia & histologia , Cabras/embriologia , Fator de Crescimento Derivado de Plaquetas/administração & dosagem , Fator de Crescimento Derivado de Plaquetas/efeitos adversos , Fertilização in vitro , Folículo OvarianoResumo
The aims of this study were to investigate the effects of different concentrations of Platelet-derived growth factor-BB (PDGF-BB) on the survival, activation, levels of ROS, and growth of goat preantral follicles enclosed in ovarian tissue. For this, ovarian fragments were cultured for 7 days in Alpha Minimum Essential Medium (α-MEM+ ) with or without PDGF-BB (0, 25, 50 and 100 ng/ml). The results showed that both the 25 ng/ml PDGF and the 50 ng/ml PDGF treatments maintained the percentage of morphologically normal follicles from day 1 to day 7. In addition, the 25 ng/ml PDGF treatment showed a significantly higher percentage of morphologically normal follicles when compared to the other treatments. At day 7, greater (P < 0.05) follicular and oocyte diameters were observed in the 25 ng/ml PDGF and the 50 ng/ml PDGF treatments when compared to the cultured control treatment. On day 7 of culture, all the treatments tested had a significant increase in the percentage of developing follicles when compared to the non-cultured control. However, the percentage of follicle activation, as well as ROS production, were similar (P < 0.05) among the treatments, irrespective of culture time. In conclusion, PDGF-BB improved, in a concentration-dependent manner, follicular survival as well as oocyte and follicular diameter after in vitro culture of goat preantral follicle-enclosed in ovarian tissue fragments.(AU)
Assuntos
Animais , Fator de Crescimento Derivado de Plaquetas/administração & dosagem , Fator de Crescimento Derivado de Plaquetas/efeitos adversos , Cabras/anatomia & histologia , Cabras/embriologia , Fertilização in vitro , Folículo OvarianoResumo
A Medicina Regenerativa visa alcançar tratamentos que acelerem as diferentes fases do processo de cicatrização em humanos e animais e a manipulação da composição dos fatores de crescimento (FC) pode melhorar ou modificar o processo de reparo e remodelação dos tecidos lesados, bem como proporcionar cicatrizes mais estéticas e funcionais. O presente estudo objetivou avaliar o efeito dos fatores peptídicos de crescimento recombinantes humanos derivado de plaquetas (PDGF- BB) e/ou de crescimento endotelial e/ou vascular (VEGF165) em células-tronco mesenquimais do tecido adiposo (canino) e de medula óssea (equino) e seu secretoma, e em modelo de cicatrização de pele em ratos nude Rowett. Os FC foram obtidos a partir de meios purificados por cromatografia de afinidade a heparina e caracterizados por ELISA e Western blot, e foram utilizados em tampão como veículo ou em hidrogel de alginato 2%. As células foram obtidas, caracterizadas, cultivadas e tratadas com FC e o meio condicionado foi utilizado para isolamento de exossomos. O modelo animal de cicatrização de feridas foi estabelecido em ratos nude Rowett, onde foram aplicados 3g/mL de FC sozinhos ou combinados em feridas no dorso dos animais, e após 7 dias a evolução da cicatrização foi avaliada macroscópica e histopatologicamente. O presente estudo demonstrou pela primeira vez o potencial da suplementação de rhPDGF-BB e/ou rhVEGF165 na cicatrização de feridas cutâneas em sete dias, estimulando angiogênese, reação de fibroblastos e reepitelização. O hidrogel de alginato 2% mostrou ser a melhor abordagem para ser usada como veículo para aplicações de FC in vivo em relação ao tampão veículo, mas não em ensaios in vitro. O tratamento com ambos os fatores combinados melhorou o perfil terapêutico de células-tronco mesenquimais de tecido adiposos estimulando a migração celular e a secreção de exossomos. O estudo demonstrou pela primeira vez que o tratamento com rhVEGF165 estimulou proliferação celular, o perfil angiogênico e a secreção de exossomos por células-tronco mesenquimais de medula óssea equina. Juntos os dados embasam futuras investigações sobre efeito da suplementação com FC in vitro, no conteúdo celular e molecular do secretoma de células-tronco mesenquimais, e in vivo em um modelo seguro e reprodutível, abrindo uma nova perspectiva para abordagem terapêutica livre de células para cicatrização de feridas. Palavras-chave: Cicatrização de feridas. PDGF-BB. VEGF. Células-tronco mesenquimais. Exossomos.
Regenerative Medicine aims to achieve treatments that accelerate the different stages of the healing process in both humans and animals, and the manipulation of the growth factor (GF) composition can improve or modify the repair and remodeling process of injured tissues, as well as providing more aesthetic and functional scars. The present study aimed to evaluate the effect of recombinant human platelet- derived growth factor (PDGF-BB) and/or endothelial vascular growth factor (VEGF165) on adipose (canine) and bone marrow (equine) mesenchymal stem cells and their secretome, as well as in a model of skin healing in nude rats. GF were obtained from purified media by heparin affinity chromatography and characterized by ELISA and Western blot, and were used in vehicle buffer or in 2% alginate hydrogel. Mesenchymal stem cells were obtained, characterized, cultured and treated with GF and exosomes were isolated from conditioned media. The animal model of wound healing was established in Rowett nude rats, where 3g/mL of GF were applied alone or combined in wounds on the animals' backs, and after 7 days the healing evolution was evaluated macroscopically and histopathologically. The present study demonstrated for the first time the potential of rhPDGF-BB and/or rhVEGF165 supplementation in the healing of skin wounds in seven days, stimulating angiogenesis, fibroblast reaction and re-epithelialization, but a long-term evaluation is necessary. The 2% alginate hydrogel proved to be the best approach to be used as a vehicle for GF applications in vivo in relation to the vehicle buffer, but not in vitro assays. The treatment with both factors combined improved the therapeutic profile of adipose mesenchymal stem cells by stimulating cell migration and exosome secretion. The study demonstrated for the first time that treatment with rhVEGF165 stimulated cell proliferation, angiogenic profile and secretion of exosomes by equine bone marrow mesenchymal stem cells. Together, the data support future investigations into the effect of GF supplementation in vitro, on cellular and molecular content of the mesenchymal stem cell secretome and in vivo, in a safe and reproducible model, opening a new perspective for a cell-free therapeutic approach for wound healing.
Resumo
Os objetivos do presente estudo foram investigar os efeitos de diferentes concentrações do fator de crescimento derivado de plaquetas BB (PDGF-BB) sobre a sobrevivência, ativação, níveis de especies reativas de oxigênio (EROs) e crescimento de folículos pré-antrais caprinos inclusos em tecido ovariano. Para isso, fragmentos ovarianos foram cultivados durante 7 dias em meio essencial mínimo (-MEM+) na ausência ou presença de PDGF-BB (0, 25, 50 e 100 ng/mL). Os resultados mostraram que tanto o tratamento PDGF 25 ng/mL quanto PDGF 50 ng/mL mantiveram a porcentagem de folículos morfologicamente normais do dia 1 ao dia 7. Além disso, o tratamento PDGF 25 ng/mL mostrou uma porcentagem significativamente superior de folículos morfologicamente normais quando comparadas aos outros tratamentos. No dia 7, foram observados maiores (P < 0,05) diâmetros foliculares e oocitários nos tratamentos PDGF 25 ng/mL e 50 ng/mL quando comparados ao tratamento controle cultivado. No dia 7 de cultivo, todos os tratamentos testados aumentaram significativamente a porcentagem de folículos em desenvolvimento quando comparados ao controle não cultivado. No entanto, a porcentagem de ativação folicular, bem como a produção de EROs, foram similares (P > 0,05) entre os tratamentos, independentemente do tempo de cultivo. Em conclusão, PDGF-BB melhorou, de forma concentração dependente, a sobrevivência folicular, bem como promoveu o auumento dos diâmetros do oocito e do foliculo após o cultivo in vitro de folículos pré-antrais caprinos inclusos em fragmentos de tecido ovariano.
The aims of this study were to investigate the effects of different concentrations of Platelet-derived growth factor-BB (PDGF-BB) on the survival, activation, levels of ROS and growth of goat preantral follicles enclosed in ovarian tissue. For this, ovarian fragments were cultured for 7 days in Alpha Minimum Essential Medium (-MEM+) with or without PDGF-BB (0, 25, 50 and 100 ng/mL). The results showed that both the 25 ng/mL PDGF and the 50 ng/mL PDGF treatments maintained the percentage of morphologically normal follicles from day 1 to day 7. In addition, the 25 ng/mL PDGF treatment showed a significantly higher percentage of morphologically normal follicles when compared to the other treatments. At day 7, greater (P < 0.05) follicular and oocyte diameters were observed in the 25 ng/mL PDGF and the 50 ng/mL PDGF treatments when compared to the cultured control treatment. On day 7 of culture, all treatments tested significantly increased the percentage of developing follicles when compared to the non-cultured control. However, the percentage of follicle activation, as well as ROS production, were similar (P > 0.05) among the treatments irrespective of culture time. In conclusion, PDGF-BB improved, in a concentration-dependent manner, follicular survival as well as oocyte and follicular diameter after in vitro culture of goat preantral follicle enclosed in ovarian tissue fragments.