Produção de nanobiocompósito contendo nanofibras de celulose para o cultivo de células-tronco mesenquimais e aplicação em reparo ósseo

Abstract

Cellulose nanofibers (CNFs) are natural polymers that have mechanical properties that make them very attractive for applications in the construction of polymeric matrices applied to tissue engineering. The exogenous supply of a supportive polymer matrix in combination with stem cells emerges as a promising solution for inducing stem cell differentiation and repair of bone lesions. However, although cellulose is a biocompatible material, when materials are in nanoscale become more reactive, needing to investigate its potential toxic effect to ensure safe application. The hypothesis of this work is that NFCs are cytocompatible and when used as nanobiocomposite components induce the proliferation and differentiation of human mesenchymal stem cells. The general objective of this project was to evaluate the cytocompatibility of cotton CNFs and the potential of nanobiocomposites containing CNF and chitosan in promoting the proliferation and osteogenic differentiation of human mesenchymal stem cells. In experiment 1, the physicochemical characterization of CNFs was performed and the evaluation of the potential toxic effect and induction of osteogenic differentiation of suspended CNFs in human mesenchymal stem cells extracted from the dental pulp cultivated in vitro. In experiment 2, the synthesis was performed using the solvent evaporation technique and the physical-chemical characterization of the nanobiocomposite containing CNF and chitosan. HEK293 cells were grown on this matrix to test the cytocompatibility and the nanobiocomposite's ability to promote the adhesion and proliferation of human cells. Subsequently, in experiment 3, aiming at greater stability the physicochemical properties of the nanobiocomposite were altered and, again characterized and then human mesenchymal stem cells were cultivated on the nanobiocomposite to evaluate cytocompatibility and the induction potential of osteogenic differentiation. The results of experiment 1 revealed that cotton CNFs are needle-like elongated fiber, with diameters of about 6 - 18 nm, while their length ranged from 85 to 200 nm. The cellulose characteristic bands were obtained by spectroscopy and CNFs showed negative surface charges on their surface (–10 mV). In the conditions tested, there was no difference (p>0.05) in the rates of proliferation, viability, cell cycle and extracellular SOD levels. However, exposure to CNFs for 48 h decreased the expression levels of genes related to stress and apoptosis (ATF4, DAPK1 and BAX; p<0.05). Additionally, the activity results of alkaline phosphatase (p<0.05), von Kossa and Alizarin Red demonstrated that cotton CNFs demonstrated the ability to induce osteogenic in human mesenchymal stem cells. In experiment 2, characterization results showed that CNF increased nanotopography and decreased the rates of intumescent and degradation of CNF/chitosan nanobiocomposite (p<0.05). The cytocompatibility assays, nanobiocomposite promoted cell proliferation and increased area of HEK293 cells (p<0.05), but was cytotoxic after 72 h of culture. In experiment 3, the physicochemical properties of the nanobiocomposite were altered by improving cytocompatibility, as well as presented the ability to induce osteogenic differentiation for human mesenchymal stem cells. Together, the data in this study provided new information on the cytocompatibility of cotton CNFs and nanobiocomposites containing cotton CNFs. The CNFs and nanobiocomposites have induced the proliferation and differentiation of stem cells, which opens possibilities for future applications of these biomaterials in cell therapy and bone regeneration.