Optimierung und Validierung eines ex-vivo Kultursystems für trabekulären Knochen
In dieser Arbeit wurde mit dem ZETOS System, einem neuen ex-vivo Kutursystem für trabekulären Knochen gearbeitet. Hauptziel der Arbeit war die Bestimmung der Flussrate in dem ZETOS System, bei der eine optimale physiologische Umgebung für das Wachstum explantierter Knochen gegeben ist. Bei den Unter...
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- The ZETOS system is a novel ex-vivo culturing and loading system for trabecular bone cores. The major goal of this thesis was to find the optimal flow rate for this system to yield a physiological environment for the growth of explanted bone cores. Surprisingly, the experiments showed that the bones had not been perfused instead media transport was controlled by diffusion. This result was verified by using an axial flowing system applying the same hydrostatic pressure as in the culture chamber. Therefore, the ZETOS-system should be referred to a circumfusion system rather than a perfusion system. Comparing the two culture mediums DMEM and BGJb only the bones cultured in BGJb showed an activation of osteoblasts after 26 days of culturing independent of the mechanical treatment. The relative osteoid area (Os.Ar/B.Ar) was significantly increased in loaded and not- loaded bone cores cultured in BGJb media. In DMEM there was no difference between loaded and unloaded bone cores. Since loading did not induce osteoid formation significantly, the bone surface was analyzed extensively by scanning microscopy. The uneven texture of the section surface suggests that an optimal transfer of force to the bones necessary for stimulation cannot be guaranteed using the given preparation method. Histological examination of the bone showed in all groups no sign of necrosis after 26 days of culturing. Fluorochrome double labeling as a measure of bone growth was only observed in some sections. In these samples, however, it was possible to measure a physiological mineral aposition rate (MAR) as a parameter for bone growth. The MAR was comparable to published minimal bone growth rates. To prove vitality of the bones simultaneous analyses of protein and DNA synthesis are recommended for future experiments. Finally it was possible to develop two computer programs (macros) for pixel based picture analysis, that simplify the automatic histological examination of the thin sections by measuring the mineral apposition rate (MAR) and the relative osteoid surface (Os.Ar. / B.Ar.).