Bulk and interface investigations of scaffolds and tissue-engineered bones by X-ray microtomography and X-ray microdiffraction.

This review is presented of recent investigations concerning the structure of ceramic scaffolds and tissue-engineered bones and focused on two techniques based on X-ray radiation, namely microtomography (microCT) and microdiffraction. Bulk 3D information, with micro-resolution, is mainly obtained by microCT, whereas microdiffraction provides useful information on interfaces to the atomic scale, i.e. of the order of the nanometer. Since most of the reported results were obtained using synchrotron radiation, a brief description of the European Synchrotron Radiation Facility (ESRF) is presented, followed by a description of the two techniques. Then examples of microstructural investigations of scaffolds are reported together with studies on bone architecture. Finally, studies on ex vivo tissue-engineered bone and on bone microstructure in vivo are presented.

Engineering of bone using bone marrow stromal cells and a silicon-stabilized tricalcium phosphate bioceramic: evidence for a coupling between bone formation and scaffold resorption.

Resorbable porous ceramic constructs, based on silicon-stabilized tricalcium phosphate, were implanted in critical-size defects of sheep tibias, either alone or after seeding with bone marrow stromal cells (BMSC). Only BMSC-loaded ceramics displayed a progressive scaffold resorption, coincident with new bone deposition. To investigate the coupled mechanisms of bone formation and scaffold resorption, X-ray computed microtomography (muCT) with synchrotron radiation was performed on BMSC-seeded ceramic cubes. These were analyzed before and after implantation in immunodeficient mice for 2 or 6 months. With increasing implantation time, scaffold thickness significantly decreased while bone thickness increased. The muCT data evidenced that all scaffolds showed a uniform density distribution before implantation. Areas of different segregated densities were instead observed, in the same scaffolds, once seeded with cells and implanted in vivo. A detailed muX-ray diffraction analysis revealed that only in the contact areas between deposited bone and scaffold, the TCP component of the biomaterial decreased much faster than the HA component. This event did not occur at areas away from the bone surface, highlighting coupling and cell-dependency of the resorption and matrix deposition mechanisms. Moreover, in scaffolds implanted without cells, both the ceramic density and the TCP:HA ratio remained unchanged with respect to the pre-implantation analysis.

Engineered bone from bone marrow stromal cells: a structural study by an advanced x-ray microdiffraction technique.

The mechanism of mineralized matrix deposition was studied in a tissue engineering approach in which bone tissue is formed when porous ceramic constructs are loaded with bone marrow stromal cells and implanted in vivo. We investigated the local interaction between the mineral crystals of the engineered bone and the biomaterial by means of microdiffraction, using a set-up based on an x-ray waveguide. We demonstrated that the newly formed bone is well organized inside the scaffold pore, following the growth model of natural bone. Combining wide angle (WAXS) and small angle (SAXS) x-ray scattering with high spatial resolution, we were able to determine the orientation of the crystallographic c-axis inside the bone crystals, and the orientation of the mineral crystals and collagen micro-fibrils with respect to the scaffold. In this work we analysed six samples and for each of them two pores were studied in detail. Similar results were obtained in all cases but we report here only the most significant sample.

Recent advances in superhydrophobic surfaces and their relevance to biology and medicine.

By mimicking naturally occurring superhydrophobic surfaces, scientists can now realize artificial surfaces on which droplets of a few microliters of water are forced to assume an almost spherical shape and an extremely high contact angle. In recent decades, these surfaces have attracted much attention due to their technological applications for anti-wetting and self-cleaning materials. Very recently, researchers have shifted their interest to investigate whether superhydrophobic surfaces can be exploited to study biological systems. This research effort has stimulated the design and realization of new devices that allow us to actively organize, visualize and manipulate matter at both the microscale and nanoscale levels. Such precise control opens up wide applications in biomedicine, as it allows us to directly manipulate objects at the typical length scale of cells and macromolecules. This progress report focuses on recent biological and medical applications of superhydrophobicity. Particular regard is paid to those applications that involve the detection, manipulation and study of extremely small quantities of molecules, and to those that allow high throughput cell and biomaterial screening.

Synchrotron radiation microtomography of bone engineered from bone marrow stromal cells.

Osteoprogenitor cells expanded in vitro and associated with porous ceramic scaffolds have been proposed as bone substitutes. Animal models have been developed to test the efficacy of various cell populations and scaffolds in promoting bone repair. Qualitative analysis of the new bone formed within the ceramic scaffold is relatively easy by conventional histology. On the other hand, quantitative data are difficult to obtain. X-ray computed microtomography was used as a possible experimental technique to obtain quantitative data on the three-dimensional structure of newly formed bone and of remaining scaffold in implants after 8 weeks in vivo. Measurements were performed at the European Synchrotron Radiation Facility on beamline ID19 with a spatial resolution of about 5 microm. This study clearly indicates the possibility of nondestructive quantitative analysis of bone-engineered constructs. The technique appears suitable to compare different scaffolds (and possibly different cell populations) with regard to bone formation efficiency and reabsorbability of biomaterials in the immunodeficient mouse model.

X-ray micro-diffraction analysis of reconstructed bone at Zr prosthetic surface with sub-micrometre spatial resolution.

The purpose of the present investigation is to demonstrate the power of the x-ray micro-diffraction technique in biological studies. In particular the reported experiment concerns the study of the interface between a Zr prosthetic device implanted in a rat femur and the newly-formed bone, with a spatial resolution of 0.5 microm. The obtained results give interesting information on the Zr deformation and on the crystallographic phase, the grain size and the orientation of the new bone. Moreover the study reveals a marked difference in the structure of the reconstructed bone with respect to the native bone, which cannot be appreciated with other techniques.