Desenvolvimento, avaliação da toxicidade e do potencial reparador ósseo de materiais à base de fibroína ou celulose bacteriana associados à hidroxiapatita e anticorpos anti-bmp-2
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Data
2018-02-23
Autores
Coelho, Fernanda [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A engenharia tecidual voltada ao tecido ósseo tem como objetivo proporcionar uma reparação que apresente propriedades físicas e biológicas similares ao osso natural com restabelecimento adequado das suas funções. Neste contexto, materiais aloplásticos baseados em biopolímeros como a fibroína da seda (FS) e celulose bacteriana (CB) têm proporcionado a síntese de excelentes biomateriais para reparação óssea. Esses biopolímeros são biocompatíveis, possuem características mecânicas interessantes, apresentam uma lenta taxa de degradação in vivo, além da capacidade de serem modificados quimicamente. Para conferir a estes biopolímeros uma melhor bioatividade com intuito de potencializar a eficácia local da reparação óssea, o objetivo do presente estudo consistiu em sintetizar uma membrana à base de FS e outra à base de CB, sendo cada uma associada à hidroxiapatita e anticorpo anti-proteína morfogenética óssea (anti-BMP-2). Para tanto, estas membranas foram caracterizadas físico-quimicamente e, para análise do potencial destas membranas na reparação óssea, foram realizados ensaios in vitro para investigação de citotoxicidade, genotoxicidade e mutagenicidade, além de expressão gênica desses materiais. As membranas à base de CB obtiveram resultados da caracterização físico-quimicamente mais satisfatórios em relação à FS e os testes seguintes foram realizados apenas com a membrana de CB. A membrana de CB associada à hidroxiapatita não demonstrou citotoxicidade, genotoxicidade e mutagenicidade para as linhagens CHO-K1 e MC3T3-E1. Estudos de cinética de liberação de anticorpos in vitro foram realizados para confirmar a ligação de anti-BMP-2 nas membranas e o perfil de liberação dos anticorpos quando associados a anticorpos secundários conjugados com FITC. Os resultados demonstraram níveis de anticorpos detectados por até 14 dias e uma redução gradativa com o passar dos dias, reduzindo aproximadamente 70% em 7 dias e 90% em 14 dias. Referente à análise de expressão gênica dos 6 genes candidatos investigados, todos apresentaram aumento de expressão gênica, sendo os genes ALPL, RUNX2 e TNFRS11B nos primeiros dias de contato com a membrana e os genes SPP1, BGLAP e VEGF após um período de contato maior comparado ao grupo de interesse. Estudos de mineralização óssea e a atividade de fosfatase alcalina demonstraram que os grupos que apresentavam a proteína BMP-2 obtiveram uma maior resposta quando comparada ao grupo controle. Em conclusão, a membrana CB-HA-antiBMP-2 obteve resistência mecânica necessária para agir como material de preenchimento ósseo; é hidrofílica, permitindo a adesão e proliferação celular e não apresentou potencial tóxico. A adsorção dos anticorpos nas membranas foi confirmada; houve um aumento da expressão dos genes envolvidos na reparação óssea in vitro em células MC3T3-E1; houve aumento da formação de nódulos minerais e da atividade de ALP.
Tissue engineering related to the bone tissue aims to provide a repair that presents similar physical and biological properties to the natural bone with adequate restoration of its functions. In this context, alloplastic materials based on biopolymers such as silk fibroin (SF) and bacterial cellulose (BC) have provided the synthesis of excellent biomaterials for bone repair. These biopolymers are biocompatible, have interesting mechanical characteristics, have a slow rate of degradation in vivo, and the ability to be chemically modified. In order to give these biopolymers better bioactivity to potentiate the local effectiveness for bone repair, the aim of the present study was to synthesize a SF-based and BC-based membrane, each of them was associated with hydroxyapatite and antibody anti-bone morphogenetic protein (anti-BMP-2). The membranes with immobilized anti-BMP-2 are expected to be effective in capturing endogenous BMP-2, conferring a different therapeutic approach and potential use for bone tissue engineering. For this purpose, these membranes were physico-chemically characterized and, in order to analyze the potential of these membranes in bone repair, in vitro assays were performed to investigate cytotoxicity, genotoxicity, mutagenicity and gene expression of these materials. The BC-based membranes obtained physically-chemically characteristics more satisfactory than the SF, and the next tests were performed only on the BC membrane. The BC membrane associated with hydroxyapatite did not demonstrate cytotoxicity genotoxicity and mutagenicity for CHO-K1 and MC3T3-E1. In vitro antibody kinetics studies were performed to confirm the binding of anti-BMP-2 to membranes and the antibody release profile when associated with FITC-conjugated secondary antibodies. The results demonstrated antibody levels detected for up to 14 days, and a gradual reduction over the course of days, reducing approximately 70% at 7 days and 90% at 14 days. Regarding the gene expression analysis of the 6 candidate genes investigated, all presented increased gene expression, with the ALPL, RUNX2 and TNFRS11B genes in the first days of contact with the membrane, and the SPP1, BGLAP and VEGF genes after a superior contact period compared to the interest group. Studies of bone mineralization and alkaline phosphatase activity demonstrated that the groups that presented the BMP-2 protein obtained a greater response when compared to the control group. In conclusion, the BC-HA-anti-BMP-2 membrane presented the necessary mechanical resistance to act as bone filling materials; is hydrophilic, allowing adhesion and cell proliferation; and did not present toxic potential. The adsorption of the antibodies on the membranes was confirmed; there was an increase of the expression of genes involved in bone repair in vitro in MC3T3-E1 cells; there was increase of the mineral nodule formation and of the ALP activity.
Tissue engineering related to the bone tissue aims to provide a repair that presents similar physical and biological properties to the natural bone with adequate restoration of its functions. In this context, alloplastic materials based on biopolymers such as silk fibroin (SF) and bacterial cellulose (BC) have provided the synthesis of excellent biomaterials for bone repair. These biopolymers are biocompatible, have interesting mechanical characteristics, have a slow rate of degradation in vivo, and the ability to be chemically modified. In order to give these biopolymers better bioactivity to potentiate the local effectiveness for bone repair, the aim of the present study was to synthesize a SF-based and BC-based membrane, each of them was associated with hydroxyapatite and antibody anti-bone morphogenetic protein (anti-BMP-2). The membranes with immobilized anti-BMP-2 are expected to be effective in capturing endogenous BMP-2, conferring a different therapeutic approach and potential use for bone tissue engineering. For this purpose, these membranes were physico-chemically characterized and, in order to analyze the potential of these membranes in bone repair, in vitro assays were performed to investigate cytotoxicity, genotoxicity, mutagenicity and gene expression of these materials. The BC-based membranes obtained physically-chemically characteristics more satisfactory than the SF, and the next tests were performed only on the BC membrane. The BC membrane associated with hydroxyapatite did not demonstrate cytotoxicity genotoxicity and mutagenicity for CHO-K1 and MC3T3-E1. In vitro antibody kinetics studies were performed to confirm the binding of anti-BMP-2 to membranes and the antibody release profile when associated with FITC-conjugated secondary antibodies. The results demonstrated antibody levels detected for up to 14 days, and a gradual reduction over the course of days, reducing approximately 70% at 7 days and 90% at 14 days. Regarding the gene expression analysis of the 6 candidate genes investigated, all presented increased gene expression, with the ALPL, RUNX2 and TNFRS11B genes in the first days of contact with the membrane, and the SPP1, BGLAP and VEGF genes after a superior contact period compared to the interest group. Studies of bone mineralization and alkaline phosphatase activity demonstrated that the groups that presented the BMP-2 protein obtained a greater response when compared to the control group. In conclusion, the BC-HA-anti-BMP-2 membrane presented the necessary mechanical resistance to act as bone filling materials; is hydrophilic, allowing adhesion and cell proliferation; and did not present toxic potential. The adsorption of the antibodies on the membranes was confirmed; there was an increase of the expression of genes involved in bone repair in vitro in MC3T3-E1 cells; there was increase of the mineral nodule formation and of the ALP activity.
Descrição
Palavras-chave
Regeneração óssea, Anticorpos monoclonais, Toxicidade, Expressão gênica, Bone regeneration, Monoclonal antibodies, Toxicity, Gene expression