Fotoresinas biocompatíveis para microfabricação de arcabouços celulares tridimensionais

Abstract

The additive technique by Two-Photon Polymerization (P2F) has become an excellent microfabrication method among the many existing processes, as it allows the construction of three-dimensional microstructures with high spatial submicrometer resolution, with high geometric complexity and minimal use of photosensitive material. This powerful tool has attracted the interest of several areas of the science, including the area of tissue engineering with application in the manufacture of three-dimensional scaffold for cell culture. These microenvironments are designed to mimic environments in vivo and express their most important characteristics, enabling the study of the cellular behavior and the treatment of diseases with drug testing. Photosensitive resins are the raw material for the construction of scaffolds and, although there is a wide availability of these materials for biological studies, there is a great concern in making photoresist compounds biocompatible and with reduced cytotoxicity. In this work, the influence of the incorporation of semiconductor nanoparticles (NPs) of CdSe/CdS and titanium dioxide (TiO2) in photoresist based on Pentaerythritol Triacrylate (PETA) and Irgacure 369, by the use of different experimental techniques such as optical absorption spectroscopy and fluorescence spectroscopy. The degree of conversion (DC) by Raman spectroscopy, writing parameters, cytotoxicity by trypan blue exclusion method, and biomechanical properties of bone cells by Atomic Force Microscope (AFM) were also evaluated in these photoresists. The results obtained showed that there were no change in the peak of the absorption band of photoresist with the addition of NPs, therefore, the photosensitive characteristics are preserved, being suitable for the P2F process. From the fluorescence spectra (λExc= 400 nm), it was found that as the concentration of TiO2 in the photoresist increases, there was an increase in photoluminescence with a broadening of the band. Meanwhile, the incorporation of CdSe/CdS reduces the photoluminescence of the samples. The DC values of photoresists incorporated with CdSe/CdS and TiO2 were reduced from 21% to 19.5% and 17.1%, respectively. This data shows that NPs act as inhibitory agents in polymerization processes. All photoresists incorporated with NPs demonstrated acceptable biocompatibility. Notably, the sample with TiO2 showed low cytotoxicity and a high cell proliferation rate, which remained higher than the control group for 48 h. Appropriate writing parameters that raised the resolution of microstructures to sub-micrometric values were determined. Then, microgroove type scaffolds, in different geometries, were manufactured and demonstrated excellent structural conditions with an architecture compatible with the 3D design projected in nFab software, without shrinkage or deformation of the walls. Regarding biomechanical properties, human osteoblasts of the MG-63 lineage in direct contact with the framework in the circular geometry exhibited a significantly higher Young’s modulus with a 93% increase in its stiffness when compared to cells seeded on the rectangular scaffold. These results were attributed to the reorganization of the bone cell cytoskeleton influenced by the geometry and curved topography of the cellular microenvironment, which offers a greater stimulus in the biomechanical response of the cells.