Regeneração óssea induzida por scaffold β-TCP/S53P4 produzido por gel casting: estudo in vitro e in vivo
Data
2021-10-25
Autores
Amaral, Suelen Simões [Unesp]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Os materiais biocerâmicos são promissores em cirurgias de substituição óssea, tanto para aplicações odontológicas quanto ortopédicas. Estes materiais apresentam excelente compatibilidade biológica, osteocondutividade, degradação, além de exibirem atividade antimicrobiana. Neste presente trabalho, o objetivo foi avaliar in vitro a efetividade de scaffolds β-fosfato tricálcico (β-TCP) incorporado com biovidro (S53P4), produzidos por meio do processo de gel casting, na atividade celular, diferenciação osteoblástica e ação antibacteriana sobre biofilmes monotípicos, e avaliar in vivo seu efeito sobre a neoformação óssea. Os scaffolds foram confeccionados com diferentes materiais: a) β-TCP; b) β-TCP incorporado com biovidro. Na etapa in vitro, foram realizados os testes de MTT, proteína total, atividade de fosfatase alcalina, nódulos de mineralização, interação celular e expressão dos genes relacionados à osteogênese por RT-PCR. Na etapa in vivo, dois scaffolds de cada biomaterial foram implantados nas tíbias direita e esquerda de coelhos. Após 21 dias, os animais foram eutanasiados e a neoformação óssea foi avaliada por meio de análise histológica e análise histomorfométrica. Por fim, avaliou a formação de biofilmes em cepas de Candida albicans, Pseudomonas aeruginosa e Staphylococcus aureus. Os resultados in vitro, mostraram que os scaffolds β-TCP/S53P4 não apresentaram efeito citotóxico e permitiram a interação de células de aspecto alongado sobre os scaffolds. Na avaliação da proteína total os scaffolds β-TCP/S53P4 apresentaram valores mais altos, sendo significativamente maior em comparação ao β-TCP (p<0,05) no período de 7 dias. Na atividade de fosfatase alcalina, os scaffolds foram semelhantes estatisticamente (p>0,05). Na quantificação de nódulos de mineralização, os scaffolds β-TCP/S53P4 expressaram maior quantidade de nódulos de mineralização com diferença estatística significativa (p<0,05) com os scaffolds β-TCP. No período de 7 dias, os genes osteopontina (Osp), fator relacionado ao Runt (Runx2), osteocalcina (Bglap), fator estimulador de colônias de macrófagos (M-csf), prostaglandina E2 (PgE2), osteonectina (Osn) e fator de crescimento de transformador Beta (Tgf-β1) foram expressos em todos os scaffolds, sem diferença estatística entre os grupos (p>0,05). Contudo a expressão de integrina β1 (Itg β1) no scaffold β-TCP foi maior do que no scaffold β-TCP/S53P4, diferindo estatisticamente (p<0,05), houve maior expressão de colágeno I (Col-1) em β-TCP/S53P4 com diferença estatística (p<0,05). Os defeitos preenchidos por β-TCP/S53P4 tiveram maior formação e preenchimento por tecido ósseo neoformado (p<0,05) conforme a análise histomorfométrica. β-TCP/S53P4 inibiu o crescimento bacteriano e fúngico em comparação ao β-TCP (p<0,05). Desse modo, os resultados confirmam a capacidade osteogênica da cerâmica β-TCP e sugere que após a incorporação do biovidro S53P4, pode reduzir infecções microbianas. O novo biomaterial demonstrou-se um excelente material para aplicação na engenharia de tecido ósseo.
Bioceramic materials are promising in bone replacement surgeries, both for dental and orthopedic applications. These materials have excellent biological compatibility, osteoconductivity, degradation, in addition to exhibiting antimicrobial activity. The objective of this study was to investigate the effects of bioglass (S53P4) incorporated into β-tricalcium phosphate (β-TCP) scaffolds on cell activity, osteoblast differentiation and antibacterial activity in monotypic biofilms in vitro, and to evaluate the influence of this new biomaterial on in vivo bone neoformation. The scaffolds were made with different materials: a) β-TCP; b) β-TCP incorporated with bioglass. MTT test, total protein, alkaline phosphatase activity, mineralization nodules, cell interaction and expression of genes related to osteogenesis by RT-PCR were performed. Two scaffolds of each biomaterial were implanted in the right and left tibiae of rabbits. After 21 days, the animals were euthanized and bone neoformation was evaluated through histological and histomorphometric analysis. Finally, the formation of biofilms in Candida albicans, Pseudomonas aeruginosa and Staphylococcus aureus strains. The in vitro results showed that the β-TCP/S53P4 scaffolds did not show cytotoxic effect and allowed the interaction of elongated cells on the scaffolds. In the evaluation of total protein, the β-TCP/S53P4 scaffolds showed better results, being significantly higher compared to β-TCP (p<0.05) in the 7-day period. In alkaline phosphatase activity, the scaffolds were statistically similar (p>0.05). In the quantification of mineralization nodules, the β-TCP/S53P4 scaffolds expressed a greater amount of mineralization nodules with a significant difference (p<0.05) with the β-TCP scaffolds. Within 7 days, genes Osp, Runx2, Bglap, M-csf, PgE2, Osn and Tgf-β1 were expressed in all scaffolds, with no significant difference between groups (p>0.05). The expression of Itg β1 in the β-TCP scaffold was higher than in the β-TCP/S53P4 scaffold, differing statistically (p<0.05), but there was a higher expression of Col-1 in β-TCP/S53P4 with statistical difference (p<0.05). In the histological analysis, neoformed bone tissue filling the defect was observed in both scaffolds, but greater formation was observed in β-TCP/S53P4 (p<0.05). βTCP/S53P4 inhibited bacterial and fungal growth compared to β-TCP (p<0.05). Thus, the results confirm the osteogenic capacity of the β-TCP ceramic and suggest that after its incorporation into the S53P4 bioglass, it can prevent microbial infections. An excellent biomaterial for application in bone tissue engineering
Bioceramic materials are promising in bone replacement surgeries, both for dental and orthopedic applications. These materials have excellent biological compatibility, osteoconductivity, degradation, in addition to exhibiting antimicrobial activity. The objective of this study was to investigate the effects of bioglass (S53P4) incorporated into β-tricalcium phosphate (β-TCP) scaffolds on cell activity, osteoblast differentiation and antibacterial activity in monotypic biofilms in vitro, and to evaluate the influence of this new biomaterial on in vivo bone neoformation. The scaffolds were made with different materials: a) β-TCP; b) β-TCP incorporated with bioglass. MTT test, total protein, alkaline phosphatase activity, mineralization nodules, cell interaction and expression of genes related to osteogenesis by RT-PCR were performed. Two scaffolds of each biomaterial were implanted in the right and left tibiae of rabbits. After 21 days, the animals were euthanized and bone neoformation was evaluated through histological and histomorphometric analysis. Finally, the formation of biofilms in Candida albicans, Pseudomonas aeruginosa and Staphylococcus aureus strains. The in vitro results showed that the β-TCP/S53P4 scaffolds did not show cytotoxic effect and allowed the interaction of elongated cells on the scaffolds. In the evaluation of total protein, the β-TCP/S53P4 scaffolds showed better results, being significantly higher compared to β-TCP (p<0.05) in the 7-day period. In alkaline phosphatase activity, the scaffolds were statistically similar (p>0.05). In the quantification of mineralization nodules, the β-TCP/S53P4 scaffolds expressed a greater amount of mineralization nodules with a significant difference (p<0.05) with the β-TCP scaffolds. Within 7 days, genes Osp, Runx2, Bglap, M-csf, PgE2, Osn and Tgf-β1 were expressed in all scaffolds, with no significant difference between groups (p>0.05). The expression of Itg β1 in the β-TCP scaffold was higher than in the β-TCP/S53P4 scaffold, differing statistically (p<0.05), but there was a higher expression of Col-1 in β-TCP/S53P4 with statistical difference (p<0.05). In the histological analysis, neoformed bone tissue filling the defect was observed in both scaffolds, but greater formation was observed in β-TCP/S53P4 (p<0.05). βTCP/S53P4 inhibited bacterial and fungal growth compared to β-TCP (p<0.05). Thus, the results confirm the osteogenic capacity of the β-TCP ceramic and suggest that after its incorporation into the S53P4 bioglass, it can prevent microbial infections. An excellent biomaterial for application in bone tissue engineering
Descrição
Palavras-chave
Biomaterial, Biocerâmica, Biovidro, Osteogênese, Neoformação óssea, Biomaterial, Bioceramic, Bioglass, Osteogenesis, Bone neoformation