Desenvolvimento e estudo toxicológico em métodos alternativos dos compósitos à base de látex natural e fosfatos de cálcio para reparação óssea
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Data
2018-02-27
Autores
Miranda, Matheus Carlos Romeiro
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Universidade Estadual Paulista (Unesp)
Resumo
Com o aumento dos acidentes automotivos e da expectativa de vida e, consequentemente, o da população de idosos, as fraturas ósseas e a osteoporose estão mais recorrentes, incentivando assim, a busca por novos biomateriais para reparação óssea. Em vista desta problemática, o objetivo desse trabalho consistiu em estudar a influência de diferentes fases de fosfato de cálcio na liberação sustentada utilizando a membrana de latex natural (LN) como carreador para estimular a reparação óssea. Conforme os resultados de MEV/FEG, o sol-gel tratado à 400°C (CPP), 600°C (HA) e 1200°C (α-TCP), apresentaram partículas de 170-650nm, 110-620nm e 10-79nm, respectivamente. A análise de MEV/FEG da superfície das membranas apresentou partículas de CPP inclusas na superfície e no interior das membranas. No entanto, as membranas incorporadas com HA e a α-TCP apresentaram uma inclusão mais superficial. As análises DRX e FT-IR confirmaram a identidade das fases presentes nas membranas. A membrana de LN pura apresentou um valor de rugosidade 71 vezes menor que a amostra de maior rugosidade (LN+ HA). No ensaio de ângulo de contato, as amostras LN pura e LN + CPP apresentaram molhabilidade parcial, enquanto as membranas incorporadas com HA e α-TCP apresentaram molhabilidade completa. Nos ensaios de cinéticas das liberações, as membranas apresentaram uma liberação de 48% para CPP, 95% para HA e 71% para α-TCP durante 330 horas. O ensaio de atividade hemolítica apresentou 1,1% para LN pura, 0% para LN+CPP, 2,2% LN+HA e 1,1% para LN+α- TCP de hemólise. O ensaio de citotoxicidade (MTT em osteoblastos foi inconclusivo, pois enzimas contidas no látex desassociaram as células do fundo do poço da placa de cultura. No entanto, após uma lavagem das membranas LN foi realizado um ensaio de adesão, onde as células aderiram na superfície. O ensaio de toxicidade em C. elegans apresentou nas primeiras 24 horas que as amostras NRL 1dia, LN 5 dias, CPP 1 dia, CPP 5 dias, HA 1 dia, HA 5 dias, e α-TCP 1 dia não são tóxicas. No entanto, a amostra α-TCP 5 dias apresentou toxicidade significativa, onde somente 31% dos animais sobreviveram. Após 48 horas de ensaio, observou-se que as amostras NRL 1 dia, CPP 1 dia, CPP 2 dias e HA 1 dia não apresentaram toxicidade. No ensaio de embriotoxicidade em Danio rerio (Zebrafish), as amostras NRL, CPP e HA apresentaram uma taxa de sobrevivência de aproximadamente 60%, que foi somente 16% menor que controle. No entanto, a amostra α-TCP a partir de 72 horas, apresentou um aumento de 24,82% na mortalidade dos embriões. Estes resultados indicaram que as membranas de LN puras e incorporadas com CPP e HA tem potencial para aplicações biomédicas em reparo ósseo.
The increase in car accidents and life expectancy, and consequently, elderly population, bone fractures and osteoporosis are more frequent, thus encouraging the search for new biomaterials for bone repair. In view of this problem, the objective of this work was to study the influence of different phases of calcium phosphate on sustained release using the natural latex (NL) membrane as carrier to stimulate bone regeneration. According to the FEG/SEM results, the sol-gel treated at 400°C (CPP), 600°C (HA) and 1200°C (α-TCP) had particles of 170-650nm, 110- 620nm and 10-79nm, respectively. FEG/SEM analysis of the membranes surface showed CPP particles included on the surface and inside the membranes. However, the incorporated membranes with HA and α-TCP had a more superficial inclusion. XRD and FT-IR analyzes confirmed the identity of the phases present in the membranes. The pure NL membrane presented a roughness value 71 times lower than the sample of greater roughness (NL + HA). In the contact angle test, the pure NL and NL + CPP samples showed partial wettability, while the incorporated membranes with HA and α-TCP showed complete wettability. In the release kinetics assays, the membranes showed a release of 48% for CPP, 95% for HA and 71% for α-TCP for 330 hours. The hemolytic activity assay presented 1.1% for pure NL, 0% for NL + CPP, 2.2% NL + HA and 1.1% for NL + α-TCP hemolysis. The cytotoxicity assay (MTT) in osteoblasts was inconclusive due enzymes contained in the latex disassociated the cells from the bottom of the well of culture plate. However, after one wash of the NL membranes, an adhesion assay was performed, where the cells adhered on surface. The toxicity test in C. elegans, showed in the first 24 hours that NL 1 day samples, NL 5 day, CPP 1 day, CPP 5 day, HA 1 day, HA 5 day, and α-TCP 1 day are non-toxics. However, the α-TCP 5 day sample presented significant toxicity, where only 31% of the animals survived. After 48 hours assay, the samples NL 1 day, CPP 1 day, CPP 2 day and HA 1 day were found to be nontoxic. Embryotoxicity assay in Danio rerio (Zebrafish) the NL, CPP and HA samples showed a survival rate of approximately 60%, which was only 16% lower than control. However, the α-TCP sample in 72 hours showed a 24.82% increase in embryo mortality. These results indicated that pure and incorporated NL membranes with CPP and HA have potential for biomedical applications in bone repair. However
The increase in car accidents and life expectancy, and consequently, elderly population, bone fractures and osteoporosis are more frequent, thus encouraging the search for new biomaterials for bone repair. In view of this problem, the objective of this work was to study the influence of different phases of calcium phosphate on sustained release using the natural latex (NL) membrane as carrier to stimulate bone regeneration. According to the FEG/SEM results, the sol-gel treated at 400°C (CPP), 600°C (HA) and 1200°C (α-TCP) had particles of 170-650nm, 110- 620nm and 10-79nm, respectively. FEG/SEM analysis of the membranes surface showed CPP particles included on the surface and inside the membranes. However, the incorporated membranes with HA and α-TCP had a more superficial inclusion. XRD and FT-IR analyzes confirmed the identity of the phases present in the membranes. The pure NL membrane presented a roughness value 71 times lower than the sample of greater roughness (NL + HA). In the contact angle test, the pure NL and NL + CPP samples showed partial wettability, while the incorporated membranes with HA and α-TCP showed complete wettability. In the release kinetics assays, the membranes showed a release of 48% for CPP, 95% for HA and 71% for α-TCP for 330 hours. The hemolytic activity assay presented 1.1% for pure NL, 0% for NL + CPP, 2.2% NL + HA and 1.1% for NL + α-TCP hemolysis. The cytotoxicity assay (MTT) in osteoblasts was inconclusive due enzymes contained in the latex disassociated the cells from the bottom of the well of culture plate. However, after one wash of the NL membranes, an adhesion assay was performed, where the cells adhered on surface. The toxicity test in C. elegans, showed in the first 24 hours that NL 1 day samples, NL 5 day, CPP 1 day, CPP 5 day, HA 1 day, HA 5 day, and α-TCP 1 day are non-toxics. However, the α-TCP 5 day sample presented significant toxicity, where only 31% of the animals survived. After 48 hours assay, the samples NL 1 day, CPP 1 day, CPP 2 day and HA 1 day were found to be nontoxic. Embryotoxicity assay in Danio rerio (Zebrafish) the NL, CPP and HA samples showed a survival rate of approximately 60%, which was only 16% lower than control. However, the α-TCP sample in 72 hours showed a 24.82% increase in embryo mortality. These results indicated that pure and incorporated NL membranes with CPP and HA have potential for biomedical applications in bone repair. However
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Palavras-chave
Materiais biomédicos, Compósitos poliméricos, Látex, Fosfato de cálcio, Regeneração óssea