RESUMO
At present, one of the main limitations of three-dimensional (3D) bioprinting in tissue engineering stems from a scarcity of biomaterials tailored for specific applications. Widely used hydrogels offer an optimal printability and a suitable environment for cell growth; however, they lack the mechanical strength required for non-soft tissues, for example, cartilage, tendons, and meniscus. This work investigated the physicochemical, mechanical, and biological characteristics of a 3D-printed polycaprolactone (PCL) reinforced with multiwalled carbon nanotubes (MWCNT) and "bamboo-like" carbon nanotubes (BCNT) with the following w/w % concentrations: 0.005%, 0.01%, 0.02%, and 0.2%. The materials were analyzed with subsequent techniques: Scanning electron microscopy, nanoindentation, parallel plate rheometry, and differential scanning calorimetry. Biological evaluations were performed with normal human articular chondrocytes by confocal microscopy and proliferation assay. The study revealed that the carbon nanotubes (CNT) addition improved the rheological properties of the material by increasing the setting temperature. Moderate enhancement was observed in terms of mechanical properties. The most significant difference was noted in cell adhesion and proliferation. Pure PCL did not facilitate cell growth and mainly apoptotic cells were observed on its surface. The addition of 0.01% MWCNT resulted in enhanced adhesion and proliferation; however, the morphology of the cells remained spherical, signifying a suboptimal surface for proliferation. Interestingly, PCL reinforced with 0.02% BCNT displayed excellent facilitation of cellular adhesion and proliferation, which is uncharacteristic of pure PCL. In summary, this study investigated the potential of CNT-reinforced PCL for 3D bioprinting and tissue engineering, highlighting key physicochemical, mechanical, and biological aspects of this biomaterial.
RESUMO
Myocardial infarction (MI) is one of the most frequent causes of death in industrialized countries. Stem cells therapy seems to be very promising for regenerative medicine. Skeletal myoblasts transplantation into postinfarction scar has been shown to be effective in the failing heart but shows limitations such, e.g. cell retention and survival. We synthesized and investigated superparamagnetic iron oxide nanoparticles (SPIONs) as an agent for direct cell labeling, which can be used for stem cells imaging. High quality, monodisperse and biocompatible DMSA-coated SPIONs were obtained with thermal decomposition and subsequent ligand exchange reaction. SPIONs' presence within myoblasts was confirmed by Prussian Blue staining and inductively coupled plasma mass spectrometry (ICP-MS). SPIONs' influence on tested cells was studied by their proliferation, ageing, differentiation potential and ROS production. Cytotoxicity of obtained nanoparticles and myoblast associated apoptosis were also tested, as well as iron-related and coating-related genes expression. We examined SPIONs' impact on overexpression of two pro-angiogenic factors introduced via myoblast electroporation method. Proposed SPION-labeling was sufficient to visualize firefly luciferase-modified and SPION-labeled cells with magnetic resonance imaging (MRI) combined with bioluminescence imaging (BLI) in vivo. The obtained results demonstrated a limited SPIONs' influence on treated skeletal myoblasts, not interfering with basic cell functions.
Assuntos
Diagnóstico por Imagem/métodos , Nanopartículas de Magnetita/química , Mioblastos/metabolismo , Apoptose , Meios de Contraste/química , Compostos Férricos/química , Humanos , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Nanopartículas de Magnetita/ultraestrutura , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Espécies Reativas de Oxigênio/metabolismo , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
One of the most important but still poorly understood issues in protein chemistry is the relationship between sequence and stability of proteins. Here, we present a method for analyzing the influence of each individual residue on the foldability and stability of an entire protein. A randomly mutated library of the crystallizable fragment of human immunoglobulin G class 1 (IgG1-Fc) was expressed on the surface of yeast, followed by heat incubation at 79°C and selection of stable variants that still bound to structurally specific ligands. High throughput sequencing allowed comparison of the mutation rate between the starting and selected library pools, enabling the generation of a stability landscape for the entire CH3 domain of human IgG1 at single residue resolution. Its quality was analyzed with respect to (i) the structure of IgG1-Fc, (ii) evolutionarily conserved positions and (iii) in silico calculations of the energy of unfolding of all variants in comparison with the wild-type protein. In addition, this new experimental approach allowed the assignment of functional epitopes of structurally specific ligands used for selection [Fc γ-receptor I (CD64) and anti-human CH2 domain antibody] to distinct binding regions in the CH2 domain.