Human bone marrow stroma stem cell distribution in calcium carbonate scaffolds using two different seeding methods.

OBJECTIVE: The aim of this study was to develop a method for the determination of the three-dimensional (3D) distribution of cells in mineralized scaffolds and to compare the effect of two different methods of cell seeding of human bone marrow stroma cells (hBMSCs) in long-term cultures.

MATERIALS AND METHODS: hBMSCs were seeded into CaCO(3) scaffolds by droplet seeding using culture medium with and without the addition of fibrin. After 2, 7, 14, and 21 days of culture, the constructs were embedded into methylmethacrylate and serially sectioned using undecalcified thick section technology. Sections were serially scanned from the surface to the bottom of the scaffolds and DAPI-stained cells were automatically counted in each section using structured illumination fluorescence microscopy (FM) with serial optical sectioning and image analysis software.

RESULTS: The data showed that the seeding efficiency was significantly higher in the scaffolds seeded with the addition of fibrin. Moreover, the number of cells increased to higher levels and remained higher for longer periods with the use of the fibrin matrix, whereas cells seeded in the medium suspension exhibited a sharp decrease after the first week of cultivation. There were distinct differences in the 3D cell distribution between the center and the periphery of the scaffolds. The use of a fibrin matrix was associated with a more uniform cell distribution 1 and 2 weeks after seeding in different levels (center vs. periphery: P>0.05), whereas cells in the medium solution group accumulated at the periphery of the scaffolds.

CONCLUSIONS: In conclusion, automated serial optical sectioning using structured illumination FM can assess cell numbers and the 3D distribution of hBMSCs in mineralized scaffolds. This allows for a detailed analysis of the effect of different in vitro procedures used for cell seeding. The use of fibrin during seeding increases seeding efficiency and enhances both proliferation and cell survival in the central parts of the scaffolds.