Lyophilized platelet-rich plasma associated with nanohydroxyapatite in cell performance and bone regeneration

Author(s)

Marcel Rodrigues Ferreira

• Document type: Doctoral Thesis
• Press: Botucatu. 2023-03-10.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2023-01-20
• Advisor: Willian Fernando Zambuzzi

Abstract

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. (AU)

• FAPESP's process: 18/05731-7 - Lyophilized PRP associated with nano-hydroxyapatite in cell performance and bone regeneration
• Grantee: Marcel Rodrigues Ferreira
• Support Opportunities: Scholarships in Brazil - Doctorate