RESUMEN
The knowledge of the mechanical properties is the starting point to study the mechanobiology of mesenchymal stem cells and to understand the relationships linking biophysical stimuli to the cellular differentiation process. In experimental biology, Atomic Force Microscopy (AFM) is a common technique for measuring these mechanical properties. In this paper we present an alternative approach for extracting common mechanical parameters, such as the Young's modulus of cell components, starting from AFM nanoindentation measurements conducted on human mesenchymal stem cells. In a virtual environment, a geometrical model of a stem cell was converted in a highly deformable Coarse-Grained Elastic Network Model (CG-ENM) to reproduce the real AFM experiment and retrieve the related force-indentation curve. An ad-hoc optimization algorithm perturbed the local stiffness values of the springs, subdivided in several functional regions, until the computed force-indentation curve replicated the experimental one. After this curve matching, the extraction of global Young's moduli was performed for different stem cell samples. The algorithm was capable to distinguish the material properties of different subcellular components such as the cell cortex and the cytoskeleton. The numerical results predicted with the elastic network model were then compared to those obtained from hertzian contact theory and Finite Element Method (FEM) for the same case studies, showing an optimal agreement and a highly reduced computational cost. The proposed simulation flow seems to be an accurate, fast and stable method for understanding the mechanical behavior of soft biological materials, even for subcellular levels of detail. Moreover, the elastic network modelling allows shortening the computational times to approximately 33% of the time required by a traditional FEM simulation performed using elements with size comparable to that of springs.
Asunto(s)
Células Madre Mesenquimatosas , Simulación por Computador , Módulo de Elasticidad , Humanos , Fenómenos Mecánicos , Microscopía de Fuerza AtómicaRESUMEN
Herein we describe an interfacial local drug delivery system for bone morphogenetic protein 2 (BMP-2) based on coatings of polyelectrolyte complex (PEC) nanoparticles (NP). The application horizon is the functionalization of bone substituting materials (BSM) used for the therapy of systemic bone diseases. Nanoparticular ternary complexes of cationic and anionic polysaccharides and BMP-2 or two further model proteins, respectively, were prepared in dependence of the molar mixing ratio, pH value and of the cationic polysaccharide. As further proteins chymotrypsin (CHY) and papain (PAP) were selected, which served as model proteins for BMP-2 due to similar isoelectric points and molecular weights. As charged polysaccharides ethylenediamine modified cellulose (EDAC) and trimethylammonium modified cellulose (PQ10) were combined with cellulose sulphatesulfate (CS). Mixing diluted cationic and anionic polysaccharide and protein solutions according to a slight either anionic or cationic excess charge colloidal ternary dispersions formed, which were cast onto germanium model substrates by water evaporation. Dynamic light scattering (DLS) demonstrated, that these dispersions were colloidally stable for at least one week. Fourier Transform Infrared (FTIR) showed, that the cast protein loaded PEC NP coatings were irreversibly adhesive at the model substrate in contact to HEPES buffer and solely CHY, PAP and BMP-2 were released within long-term time scale. Advantageously, out of the three proteins BMP-2 showed the smallest initial burst and the slowest release kinetics and around 25% of the initial BMP-2 content were released within 14days. Released BMP-2 showed significant activity in the myoblast cells indicating the ability to regulate the formation of new bone. Therefore, BMP-2 loaded PEC NP are suggested as novel promising tool for the functionalization of BSM used for the therapy of systemic bone diseases.
Asunto(s)
Proteína Morfogenética Ósea 2/química , Coloides/química , Polisacáridos/química , Animales , Aniones , Enfermedades Óseas/terapia , Huesos/metabolismo , Cationes , Adhesión Celular , Celulosa/química , Quimotripsina/química , Dicroismo Circular , Electrólitos , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Luz , Ensayo de Materiales , Ratones , Microscopía Electrónica de Rastreo , Peso Molecular , Nanopartículas/química , Papaína/química , Dispersión de Radiación , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
A major cause of implant failure in skeletal tissues is failure of osseointegration, often due to lack of adhesion of cells to the titanium (Ti) alloy interface. Since arginine-glycine-aspartic acid (RGD)-containing peptides have been shown to regulate osteoblast adhesion, we tested the hypothesis that, bound to a Ti surface, these peptides would promote osteoblasts differentiation, while at the same time inhibit apoptosis. RGDS and RGES (control) peptides were covalently linked to Ti discs using an APTS linker. While the grafting of both RGDS and RGES significantly increased Ti surface roughness, contact angle analysis showed that APTS significantly increased the surface hydrophobicity; when the peptides were tethered to Ti, this was reduced. To evaluate attachment, MC3T3-E1 osteoblast cells were grown on these discs. Significantly more cells attached to the Ti-grafted RGDS then the Ti-grafted RGES control. Furthermore, expression of the osteoblasts phenotype was significantly enhanced on the Ti-grafted RGDS surface. When cells attached to the Ti-grafted RGDS were challenged with staurosporine, an apoptogen, there was significant inhibition of apoptosis; in contrast, osteoblasts adherent to the Ti-grafted RGES were killed. It is concluded that RGD-containing peptides covalently bonded to Ti promotes osteoblasts attachment and survival with minimal changes to the surface of the alloy. Therefore, such modifications to Ti would have the potential to promote osseointegration in vivo.
Asunto(s)
Aleaciones , Apoptosis , Diferenciación Celular , Oligopéptidos , Osteoblastos/ultraestructura , Titanio , Animales , Adhesión Celular , Línea Celular , Supervivencia Celular , Materiales Biocompatibles Revestidos , Ratones , Microscopía Electrónica de Rastreo , Propiedades de SuperficieRESUMEN
The major objective of this work was to attach bone cells to a deformable surface for the effective transmission of force. We functionalized a silastic membrane and treated it with 3-aminopropyltriethoxysilane (APTS). A minimal RGD peptide was then covalently linked to the aminated surface. MC3T3-E1 osteoblast-like cells were cultured on the arginine-glycine-aspartic acid (RGD)-treated membrane for 3-15 days and cell attachment and proliferation was evaluated. We observed that cells were immediately bound to the membrane and proliferated. After 8 days on the material surface, osteoblasts exhibited high levels of ALP staining, indicating that the cells were undergoing maturation. Alizarin red staining and Fourier transform infrared (FTIR) analysis showed that the mineral formed by the cells was a biological apatite. The second objective was to apply a mechanical force to cells cultured on the modified silicone membrane. Dynamic equibiaxial strain, 2% magnitude, and a 0.25-Hz frequency were applied to bone cells for 2 h. Osteoblasts elicited increased phalloidin fluorescence, suggesting that there was reorganization of the cytoskeleton. Furthermore, the applied strain elicited increased expression of the alpha(v)beta3 integrin receptor. We concluded that the covalent binding of RGD peptides to a silicone membrane provides a compatible surface for the attachment and subsequent differentiation of osteoblasts. Moreover, the engineered surface transduces applied mechanical forces directly to the adherent cells via integrin receptors.