Interação de proteínas em superfícies de filmes finos de TiO2 e sua resposta biológica
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Data
2019-03-28
Autores
Gomes, Orisson Ponce
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Universidade Estadual Paulista (Unesp)
Resumo
O dióxido de titânio (TiO2) nanoestruturado tem sido empregado como modificador de superfície em implantes médicos, promovendo melhoria na resistência à biocorrosão do material e aumento da bioatividade, apresentando resultados promissores na interação com o tecido vivo. Apesar da biocompatibilidade deste óxido ser reconhecida, ainda existem muitos aspectos dos mecanismos de adesão entre as proteínas e a superfície do material que não são totalmente compreendidos. Quimicamente, a superfície desses óxidos é principalmente terminada por grupos -OH que podem ser prontamente funcionalizados. Essa funcionalização química ou simplesmente uma alteração física da superfície do material, pode melhorar a interação do óxido com o ambiente biológico. Além disso, dióxido de titânio pode ter o número de grupos hidroxila aumentado por indução física sob exposição a luz ultravioleta. Embora os grupos gerados por esse processo sejam termodinamicamente menos estáveis, o número maior de sítios ativos disponíveis para serem ligados às moléculas orgânicas pode resultar em uma funcionalização mais eficiente. Nesse trabalho são apresentados os estudos conformacionais de adsorção de três moléculas bifuncionais diferentes, ácido 3-mercaptopropiônico (MPA), 3-aminopropiltrimetoxisilano (APTMS) e ácido 3-(4-aminofenil)propiônico (APPA), que atuam como espaçadoras no processo de imobilização de proteína nas superfícies fisicamente hidroxiladas dos filmes de TiO2. Os filmes de TiO2 foram crescidos sobre substratos de titânio metálico grau IV através da técnica Reactive RF Magnetron Sputtering. Resultados obtidos por XPS mostraram maior eficiência na imobilização da molécula de APTMS sobre a superfície hidroxilada de TiO2 em relação às moléculas de MPA e APPA. Além disso, a molécula de APTMS também apresentou resultados satisfatórios na imobilização da proteína albumina de soro bovino (BSA). Resultados referentes aos testes de viabilidade, adesão celular e expressão dos genes relacionados à adesão celular após contato direto com células ósseas mostraram melhor interação da superfície modificada pela imobilização da BSA quando intermediada pela molécula de APTMS. As células ósseas em contato direto com essa superfície apresentaram maior expressão dos genes relacionados ao estágio inicial da diferenciação osteoblástica com relação às células em contato direto com a superfície cuja albumina foi imobilizada diretamente à superfície de TiO2. Resultados obtidos através da zimografia também corroboraram com os apresentados por expressão gênica, dado o envolvimento das MMP2 e MMP9 no processo de diferenciação osteoblástica.
Nanostructured titanium dioxide (TiO2) has been used as a surface modifier in medical implants, promoting improved biocorrosion resistance of the material and increased bioactivity, presenting promising results in the interaction with the living tissue. Despite the biocompatibility of this oxide is recognized, there are still many aspects of the adhesion mechanisms between the proteins and the material surface that are not completely understood. Chemically, the surface of these oxides is mainly terminated by -OH groups which can be readily functionalized. This chemical functionalization or simply a physical modification of the material surface can improve the interaction between the oxide and the biological environment. In addition, titanium dioxide may have the number of hydroxyl groups increased by physical induction under exposure to ultraviolet light. Although this hydroxylation process is less stable, it is possible to increase the number of active sites for a more efficient functionalization. Although the groups generated by this process are thermodynamically less stable, the greater number of active sites available to bound to organic molecules can result in a more efficient functionalization. In this work, the adsorption conformational studies of three different bifunctional molecules, 3-mercaptopropionic acid (MPA), 3-aminopropyltrimethoxysilane (APTMS) and 3-(4-aminophenil) propionic acid (APPA), are presented. These molecules act as spacers in the protein immobilization process on the physically hydroxylated surfaces of TiO2 films. The TiO2 films were grown on grade IV metallic titanium substrates by Reactive RF Magnetron Sputtering technique. XPS results showed greater efficiency in the immobilization of APTMS molecule on the hydroxylated surface of TiO2 in comparison to MPA and APPA molecules. In addition, the APTMS molecule also showed satisfactory results in the immobilization of bovine serum albumin (BSA) protein. Results regarding viability tests, cell adhesion and expression of genes related to cell adhesion after direct contact with bone cells showed better interaction of the surface modified by the immobilization of BSA when intermediated by APTMS molecule. Bone cells in direct contact with this surface showed greater expression of the genes related to the initial stage of osteoblastic differentiation with respect to cells in direct contact with the surface whose albumin was immobilized directly on TiO2 surface. Results obtained by zymography also corroborated with those presented by gene expression, given the involvement of MMP2 and MMP9 in the process of osteoblastic differentiation.
Nanostructured titanium dioxide (TiO2) has been used as a surface modifier in medical implants, promoting improved biocorrosion resistance of the material and increased bioactivity, presenting promising results in the interaction with the living tissue. Despite the biocompatibility of this oxide is recognized, there are still many aspects of the adhesion mechanisms between the proteins and the material surface that are not completely understood. Chemically, the surface of these oxides is mainly terminated by -OH groups which can be readily functionalized. This chemical functionalization or simply a physical modification of the material surface can improve the interaction between the oxide and the biological environment. In addition, titanium dioxide may have the number of hydroxyl groups increased by physical induction under exposure to ultraviolet light. Although this hydroxylation process is less stable, it is possible to increase the number of active sites for a more efficient functionalization. Although the groups generated by this process are thermodynamically less stable, the greater number of active sites available to bound to organic molecules can result in a more efficient functionalization. In this work, the adsorption conformational studies of three different bifunctional molecules, 3-mercaptopropionic acid (MPA), 3-aminopropyltrimethoxysilane (APTMS) and 3-(4-aminophenil) propionic acid (APPA), are presented. These molecules act as spacers in the protein immobilization process on the physically hydroxylated surfaces of TiO2 films. The TiO2 films were grown on grade IV metallic titanium substrates by Reactive RF Magnetron Sputtering technique. XPS results showed greater efficiency in the immobilization of APTMS molecule on the hydroxylated surface of TiO2 in comparison to MPA and APPA molecules. In addition, the APTMS molecule also showed satisfactory results in the immobilization of bovine serum albumin (BSA) protein. Results regarding viability tests, cell adhesion and expression of genes related to cell adhesion after direct contact with bone cells showed better interaction of the surface modified by the immobilization of BSA when intermediated by APTMS molecule. Bone cells in direct contact with this surface showed greater expression of the genes related to the initial stage of osteoblastic differentiation with respect to cells in direct contact with the surface whose albumin was immobilized directly on TiO2 surface. Results obtained by zymography also corroborated with those presented by gene expression, given the involvement of MMP2 and MMP9 in the process of osteoblastic differentiation.
Descrição
Palavras-chave
Sputtering, Dióxido de titânio, Hidroxilação, Funcionalização, Biomateriais, Proteínas, TiO2, Hydroxylation, Functionalization, Biomaterials, Proteins