Plasma-rico em plaquetas liofilizado associado à nanohidroxiapatita na performance celular e regeneração óssea
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Data
2023-01-20
Autores
Ferreira, Marcel Rodrigues [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Lesões ósseas constituem um dos problemas mais prementes na área médico-odontológica. Embora o tecido ósseo seja um tecido altamente dinâmico com boa capacidade de se regenerar, em caso de lesões extensas faz-se necessário o uso de biomateriais que permitam uma boa regeneração tecidual. Até o presente, não foi desenvolvido nenhum material sintético que reúna as propriedades e capacidades ofertadas pelo material autógeno, conhecidamente como material ideal. Dentre as diversas terapias propostas, a associação de biomateriais em escala nanométrica, como hidroxiapatita e/ou fosfato tricálcio, vem apresentando resultados positivos no processo de regeneração do tecido ósseo. Outra vertente amplamente utilizada é o uso de plasma rico em plaqueta (PRP), obtido do próprio paciente, no tratamento destas lesões. Baseando-nos neste prévio conhecimento sobre o potencial de ambas as terapias, o objetivo desta tese é a associação de hidroxiapatita em escala nanométrica (nanoHAp) e/ou fosfato tricálcio (βTCP) ao PRP liofilizado para a obtenção de um produto capaz de melhorar a regeneração óssea e por conseguinte a qualidade de vida do paciente. A experiência prévia do nosso grupo foi utilizada para uma caracterização do impacto deste compósito nas vias de sinalização clássicas relacionadas a processos de diferenciação de osteoblastos e regeneração do tecido ósseo através de ensaio in vitro. Embora o tema desta tese tenha sido amplamente explorado na literatura nossa motivação é justamente conhecer os aspectos celulares e moleculares que regem sua atuação durante os mecanismos de regeneração, além de propor protocolos prévios de, como resultado da proposição de novos métodos metodológicos alternativas na prática clínica, facilitando sua aplicação em lesões do tecido ósseo, amparado por um repertório molecular que justifica sua escolha e sua atuação, parâmetros altamente necessários para interagir com o setor produtivo. Foi utilizado o conhecimento em bioinformática para iniciar a compreensão do efeito de miRNAs presentes em micropartículas de plaquetas (PMPs) na osteogênese, adicionando uma nova faceta ao panorama do tratamento autólogos de PRP no tecido ósseo. Na sequência foi iniciada a padronização da obtenção PRP e sua liofilização, realizando a caracterização físico-química utilizando espectroscopia de infravermelho e molecular com ELISA. A performance celular foi avaliada em pré-osteoblastos (MC3T3-E1) e mesenquimais (MS) com ensaios de citotoxicidade e utilizando expressão gênica relativa em modelo de tratamento por 24 h e 7 dias, avaliando períodos iniciais da diferenciação osteoblástica. A síntese e caracterização físico-química de HAp e TCP foi realizada. Utilizando alginato realizamos a obtenção de hidrogéis associando PRP e HAp ou TCP e avaliamos a performance de células MC3T3-E1 e Raw 264.7 submetidas a meio condicionado por 24 h pelos hidrogéis. No geral, nossos resultados sinalizam que o PRP liofilizado afeta o desempenho dos osteoblastos de maneira semelhante ao PRP líquido quando avaliado em vias clássicas. Também foi possível concluir que a associação do PRP com os fosfatos de cálcio avaliados foi possível através da fabricação de hidrogéis de alginato de sódio.
Bone injuries are one of the most pressing problems in the medical-dental field. Although bone tissue is a highly dynamic tissue with an excellent ability to regenerate, biomaterials are necessary to allow good tissue regeneration in more severe injuries. No synthetic material has been developed that brings together the properties and capabilities offered by the autogenous material, known as the ideal material. Among the various proposed therapies, the association of biomaterials on a nanometric scale, such as hydroxyapatite and/or tricalcium phosphate, has shown positive results in the bone tissue regeneration process. Another widely used aspect is the use of platelet-rich plasma (PRP), obtained from the patient, to treat these lesions. Based on this prior knowledge about the potential of both therapies, the objective of this thesis is the association of hydroxyapatite on a nanometric scale (nanoHAp) and/or tricalcium phosphate (TCP) to lyophilized PRP to obtain a product capable of impacting the bone regeneration and therefore the patient's quality of life. We used our group's previous experience to characterize the impact of this composite on classical signaling pathways related to osteoblast differentiation processes and bone tissue regeneration through an in vitro assay. Although the theme of this thesis was widely explored in the literature, our motivation is precisely to know cellular and molecular aspects that govern its performance during regeneration mechanisms, in addition to proposing previous synthesis/combination protocols, as an outcome to propose new methodological alternatives to clinical practice, facilitating its application in bone tissue injuries, supported by a molecular repertoire that justifies its choice and performance, essential parameters for interface with the productive sector. At first, we used knowledge in bioinformatics to understand the effect of miRNAs present in platelet microparticles (PMPs) on osteogenesis, adding a new facet to the panorama of autologous PRP treatment in bone tissue. Next, we started the standardization of PRP obtaining and its lyophilization, performing the physical-chemical characterization using infrared and molecular spectroscopy with ELISA. Cellular performance was evaluated in MC3T3-E1 and mesenchymal (MS) cells with cytotoxicity assays and using relative gene expression in a 24-h and 7-day treatment model, evaluating early periods of osteoblastic differentiation. We started the synthesis and characterization of HAp and TCP. Using alginate, we carried out the construction of hydrogels associating PRP and HAp or TCP, and we evaluated the performance of MC3T3-E1, and Raw 264.7 cells submitted to medium conditioned for 24 h by the hydrogels. Our results demonstrate no cytotoxicity of our materials, supporting the following tests in vitro 3D and in vivo models.
Bone injuries are one of the most pressing problems in the medical-dental field. Although bone tissue is a highly dynamic tissue with an excellent ability to regenerate, biomaterials are necessary to allow good tissue regeneration in more severe injuries. No synthetic material has been developed that brings together the properties and capabilities offered by the autogenous material, known as the ideal material. Among the various proposed therapies, the association of biomaterials on a nanometric scale, such as hydroxyapatite and/or tricalcium phosphate, has shown positive results in the bone tissue regeneration process. Another widely used aspect is the use of platelet-rich plasma (PRP), obtained from the patient, to treat these lesions. Based on this prior knowledge about the potential of both therapies, the objective of this thesis is the association of hydroxyapatite on a nanometric scale (nanoHAp) and/or tricalcium phosphate (TCP) to lyophilized PRP to obtain a product capable of impacting the bone regeneration and therefore the patient's quality of life. We used our group's previous experience to characterize the impact of this composite on classical signaling pathways related to osteoblast differentiation processes and bone tissue regeneration through an in vitro assay. Although the theme of this thesis was widely explored in the literature, our motivation is precisely to know cellular and molecular aspects that govern its performance during regeneration mechanisms, in addition to proposing previous synthesis/combination protocols, as an outcome to propose new methodological alternatives to clinical practice, facilitating its application in bone tissue injuries, supported by a molecular repertoire that justifies its choice and performance, essential parameters for interface with the productive sector. At first, we used knowledge in bioinformatics to understand the effect of miRNAs present in platelet microparticles (PMPs) on osteogenesis, adding a new facet to the panorama of autologous PRP treatment in bone tissue. Next, we started the standardization of PRP obtaining and its lyophilization, performing the physical-chemical characterization using infrared and molecular spectroscopy with ELISA. Cellular performance was evaluated in MC3T3-E1 and mesenchymal (MS) cells with cytotoxicity assays and using relative gene expression in a 24-h and 7-day treatment model, evaluating early periods of osteoblastic differentiation. We started the synthesis and characterization of HAp and TCP. Using alginate, we carried out the construction of hydrogels associating PRP and HAp or TCP, and we evaluated the performance of MC3T3-E1, and Raw 264.7 cells submitted to medium conditioned for 24 h by the hydrogels. Our results demonstrate no cytotoxicity of our materials, supporting the following tests in vitro 3D and in vivo models.
Descrição
Palavras-chave
Tecido ósseo, Plasma-rico em plaquetas, Biomateriais, Bioinformática, Engenharia de tecidos