Application of an X-ray microscopy technique to evaluate tissue-engineered bone-scaffold constructs

Elemental composition and structural analysis are important considerations in evaluating osseous matrix formation in tissue-engineered bone constructs. Using X-ray spectroscopy techniques, such as the X-ray scanning analytical microscope (XSAM), enables the mapping of elements with simultaneous structural imaging. The aim of this study was to evaluate the feasibility of this microscopy technique to analyze tissue-engineered samples. Fused deposition modeling (FDM), a rapid prototyping technology, was used to fabricate 3-D scaffolds made of polycaprolactone (PCL)–hydroxylapatite (HA) (90/10 wt.%). The scaffolds had a regular architecture and the honeycomb-like pores were fully interconnected with a total matrix porosity of 70%. Biopsies of human calvarial corticocancellous bone were harvested and a primary explant system of the morcellized grafts was established within the PCL–HA constructs.

Tissue constructs were cultured in vitro for 3 weeks and then implanted into the back of Balb C nude mice. Grafts were explanted after 17 weeks and tissue formation was assessed via XSAM and CT scan and histology.

Outgrowth from osteoblasts from the bone chips started after 2 weeks in culture, with cells migrating radially into the composite constructs. Osteocalcin levels showed an increasing tendency during the 3-week culture period. After 17 weeks in vivo, areas of ectopic bone formation could be detected throughout the whole construct, compared to the control implants where only fibrous tissue formation was present. In the XSAM, calcium- and phosphorus-enriched bands presented a relatively even distribution with some dense spots. Quantitative elemental analysis revealed an average X-ray intensity of 131 cps for calcium in a mapped area of 2 mm². Trichrome Goldner staining showed good vascularization as well as irregular osteoid formation and mineralization of the newly formed woven bone-like tissue.

In this study, we were able to show that the XSAM is a useful analytical technique which enabled us to trace elements simultaneously and provides structural image with mapping information in tissue-engineered samples.