Biological characterization of hemostatic agents – in vitro studies
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2020-02-05
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A hemorragia é uma das principais causas de mortalidade evitável e o reconhecimento deste facto levou a grandes avanços no âmbito do controlo da hemóstase, assim como no crescimento de agentes hemostáticos disponíveis em ambientes clínicos, que são uma opção terapêutica bastante importante para o tratamento de hemorragias quando os processos fisiológicos demonstram ser ineficientes. No entanto, como estes agentes hemostáticos não passam por uma avaliação completa e regular, assim como outros biomateriais ou fármacos, atualmente não existe uma abordagem sistemática para a aquisição e documentação do uso destes agentes em contextos clínicos humanos. Assim, este trabalho tem como objetivo abordar a caracterização biológica de agentes hemostáticos à base de gelatina clinicamente disponíveis, através de estudos in vitro com várias linhas celulares humanas. As células fibroblásticas e as células osteoblásticas foram cultivadas por 24 horas em meio de crescimento (a-MEM com 10% de soro bovino fetal (SBF), 100 UI/mL de penicilina, 100 UI/mL de estreptomicina e 2,5 µg/mL de anfotericina B); após este tempo foram adicionados os extratos, previamente diluídos em a-MEM, a 50%, 25% e 12,5%. Células sem extratos foram semeadas como grupos de controlo negativo. As culturas celulares foram então avaliadas quanto à morfologia celular, proliferação celular, viabilidade celular, atividade metabólica e atividade da fosfatase alcalina, bem como histoquímica, especificamente coloração da fosfatase alcalina e conteúdo de colágenio, em diferentes timepoints. De um modo geral, os resultados não mostraram citotoxicidade significativa em nenhuma das células, exibindo de facto resultados semelhantes às células do controlo; no entanto, houve algumas diferenças nos ensaios realizados que poderiam demonstrar que, embora as esponjas não afetem a proliferação celular, elas podem ter algum efeito na sua viabilidade. Além disso, também foi possível detetar que esponjas diferentes obtiveram melhores resultados para cada tipo celular. Por estes motivos, são necessários mais estudos para elucidar os mecanismos subjacentes a estes resultados.
Hemorrhage is a leading cause of preventable mortality and recognition of this has led to progressive advances in hemostasis control and in a growth of clinically available topical hemostatic agents, which are an important therapeutic option for the management of bleeding, in which physiological processes are inefficient. However, because these hemostatic products do not undergo a thorough regulatory evaluation as do other biomaterials or substances, there is currently no systematic approach for the acquisition and documentation of use of these agents in human clinical settings. As such, this work aims to address the biological characterization of clinically available gelatin-based hemostatic agents, through in vitro studies with various human cell lines. Fibroblastic cells and osteoblastic-like cells were cultured for 24 hours in growth medium (a-MEM with 10% FBS, 100 UI/mL penicilin, 100 UI/mL streptomycin and 2,5 µg/mL amphotericin B) after which the leachables, previously diluted in a-MEM, were added at 50%, 25% and 12,5%. Cells without leachables were seeded as negative control groups. The cell cultures were then evaluated on cellular morphology, cell proliferation, cell viability, metabolic activity and alkaline phosphatase activity, as well as histochemistry, specifically alkaline phosphatase staining and collagen content, at different timepoints. Generally speaking, the results didn’t show any significant cytotoxicity in any of the cells, exhibiting in fact similar results to the control cells; however, there were some differences in the assays performed that could demonstrate that while the sponges’ may not affect cell proliferation, they could have some effect on their viability. Aside from this, it could also be detected that different sponges had better results for each cell type. For these reasons, further studies are needed in order to elucidate the mechanisms underlying these results.
Hemorrhage is a leading cause of preventable mortality and recognition of this has led to progressive advances in hemostasis control and in a growth of clinically available topical hemostatic agents, which are an important therapeutic option for the management of bleeding, in which physiological processes are inefficient. However, because these hemostatic products do not undergo a thorough regulatory evaluation as do other biomaterials or substances, there is currently no systematic approach for the acquisition and documentation of use of these agents in human clinical settings. As such, this work aims to address the biological characterization of clinically available gelatin-based hemostatic agents, through in vitro studies with various human cell lines. Fibroblastic cells and osteoblastic-like cells were cultured for 24 hours in growth medium (a-MEM with 10% FBS, 100 UI/mL penicilin, 100 UI/mL streptomycin and 2,5 µg/mL amphotericin B) after which the leachables, previously diluted in a-MEM, were added at 50%, 25% and 12,5%. Cells without leachables were seeded as negative control groups. The cell cultures were then evaluated on cellular morphology, cell proliferation, cell viability, metabolic activity and alkaline phosphatase activity, as well as histochemistry, specifically alkaline phosphatase staining and collagen content, at different timepoints. Generally speaking, the results didn’t show any significant cytotoxicity in any of the cells, exhibiting in fact similar results to the control cells; however, there were some differences in the assays performed that could demonstrate that while the sponges’ may not affect cell proliferation, they could have some effect on their viability. Aside from this, it could also be detected that different sponges had better results for each cell type. For these reasons, further studies are needed in order to elucidate the mechanisms underlying these results.
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Master’s degree in Clinical Laboratory Biology