Evaluation of bone scaffolds by micro-CT.

This paper reviews the possibilities offered by X-ray micro-CT in bone tissue engineering. This technique provides a fast, nondestructive, and 3D quantification of bone scaffolds, bone ingrowth, and microvascularization. Synchrotron radiation absorption and phase micro-CT offer additional advantages to image newly formed bone in bioceramic scaffolds and pre-bone matrix.

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.

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.

Kinetics of in vivo bone deposition by bone marrow stromal cells within a resorbable porous calcium phosphate scaffold: an X-ray computed microtomography study.

Resorbable ceramic scaffolds based on Silicon stabilized tricalcium phosphate (Si-TCP) were seeded with bone marrow stromal cells (BMSC) and ectopically implanted for 2, 4, and 6 months in immunodeficient mice. Qualitative and quantitative evaluation of the scaffold material was performed by X-ray synchrotron radiation computed microtomography (microCT) with a spatial resolution lower than 5 microm. Unique to these experiments was that microCT data were first collected on the scaffolds before implantation and then on the same scaffolds after they were seeded with BMSC, implanted in the mice and rescued after different times. Volume fraction, mean thickness and thickness distribution were evaluated for both new bone and scaffold phases as a function of the implantation time. New bone thickness increased from week 8 to week 16. Data for the implanted scaffolds were compared with those derived from the analysis of the same scaffolds prior to implantation and with data derived from 100% hydroxyapatite (HA) scaffold treated and analyzed in the same way. At variance with findings with the 100% HA scaffolds a significant variation in the density of the different Si-TCP scaffold regions in the pre- and post-implantation samples was observed. In particular a post-implantation decrease in the density of the scaffolds, together with major changes in the scaffold phase composition, was noticeable in areas adjacent to newly formed bone. Histology confirmed a better integration between new bone and scaffold in the Si-TCP composites in comparison to 100% HA composites where new bone and scaffold phases remained well distinct.