Mesenchymal Stem Cell Responses to Bone-Mimetic Electrospun Matrices Composed of Polycaprolactone, Collagen I and Nanoparticulate Hydroxyapatite

The performance of biomaterials designed for bone repair depends, in part, on the ability of the material to support the adhesion and survival of mesenchymal stem cells (MSCs). In this study, a nanofibrous bone-mimicking scaffold was electrospun from a mixture of polycaprolactone (PCL), collagen I, and hydroxyapatite (HA) nanoparticles with a dry weight ratio of 50/30/20 respectively (PCL/col/HA). The cytocompatibility of this tri-component scaffold was compared with three other scaffold formulations: 100% PCL (PCL), 100% collagen I (col), and a bi-component scaffold containing 80% PCL/20% HA (PCL/HA). Scanning electron microscopy, fluorescent live cell imaging, and MTS assays showed that MSCs adhered to the PCL, PCL/HA and PCL/col/HA scaffolds, however more rapid cell spreading and significantly greater cell proliferation was observed for MSCs on the tri-component bone-mimetic scaffolds. In contrast, the col scaffolds did not support cell spreading or survival, possibly due to the low tensile modulus of this material. PCL/col/HA scaffolds adsorbed a substantially greater quantity of the adhesive proteins, fibronectin and vitronectin, than PCL or PCL/HA following in vitro exposure to serum, or placement into rat tibiae, which may have contributed to the favorable cell responses to the tri-component substrates. In addition, cells seeded onto PCL/col/HA scaffolds showed markedly increased levels of phosphorylated FAK, a marker of integrin activation and a signaling molecule known to be important for directing cell survival and osteoblastic differentiation. Collectively these results suggest that electrospun bone-mimetic matrices serve as promising degradable substrates for bone regenerative applications

Visualizing the supramolecular assembly of collagen

Among biological macromolecules, collagen enjoys quite a peculiar status. Making up as much as a third of the protein fraction of the body it is the main responsible for the functional properties of the extracellular matrix, which can be efficiently tuned and tailored by modifying the length, volume fraction, and spatial layout of its collagen content. The supramolecular aggregates of collagen are therefore subject to be investigation by several viewpoints and at different scales, from the finest interactions of individual collagen molecules to the spatial layout of fibril bundles. As a consequence, no treatise can pretend to be exhaustive about the several techniques that can be useful in different moments and/or for different purposes. So, in this chapter, we focus only on some applications of the transmission electron microscope (TEM), of the scanning electron microscope (SEM), and of the atomic force microscope (AFM)

Bone Architecture: Collagen Structure and Calcium/Phosphorus Maps

Bone collagen structure in normal and pathological tissues is presented using techniques of thin section transmission electron microscopy and morphometry. In pathological tissue, deviations from normal fine structure are reflected in abnormal arrangements of collagen fibrils and abnormalities in fibril diameter. The relationships between these bone structural changes and the skeletal calcium/phosphorus ratio are discussed. Calcium/phosphorus ratio is measured by X-ray absorptiometry and computed microtomography

High resolution Ca/P maps ofbone architecture in 3Dsynchrotron radiation microtomographic images

The Ca/P ratio was measured in cortical bone samples from the femoral neck and tibia of different animal species, using synchrotron radiation microtomography. Use of a monoenergetic X-ray beam, as provided by the synchrotron facility, generates accurate 3D maps of the linear attenuation coefficient within the sample and hence gives the ability to map different chemical components. Also, by comparing normal and abnormal bones, i.e. osteoporotic (induced by inflammation), changes in the Ca/P ratio brought about by bone diseases can be detected. MicroCT data sets were collected at 20 and 28 keV for each bone sample and two calibration phantoms. From the 3D data sets, multiple 2D slices were reconstructed with a slice thickness of similar to30 mum. Regions of interest were defined around suitable sites and were converted to Ca/P ratios using the data collected from the test phantoms. A significant difference (p<0.001) between osteoporotics and age-matched normals at both energies was detected. Differences between different bone sites from the same animal are not significant (p>0.5) while those between the same bone sites from different animals are highly significant (p<0.001). Differences between estimates made at 20 and 28 keV are not significant (p>0.5). An important aspect is the ability to map the spatial distribution of the Ca/P ratio. (C) 2004 Elsevier Ltd. All rights reserved