Three-dimensional visualization of in vitro cultivated chondrocytes inside porous gelatine scaffolds: A tomographic approach.

Synchrotron radiation-based microcomputed tomography (SR-microCT) has become a valuable tool in the structural characterization of different types of materials, achieving volumetric details with micrometre resolution. Biomedical research dealing with porous polymeric biomaterials is one of the research fields which can benefit greatly from the use of SR-microCT. This study demonstrates that current experimental set-ups at synchrotron beamlines achieve a sufficiently high resolution in order to visualize the positions of individual cartilage cells cultivated on porous gelatine scaffolds made by a freeze-structuring technique. Depending on the processing parameters, the pore morphology of the scaffolds investigated was changed from large-pore sized but non-ordered structures to highly directional and fine pored. The cell-seeded scaffolds were stained with a combined Au/Ag stain to enhance the absorption contrast in SR-microCT. While only some cells showed enhanced absorption contrast, most cells did not show any difference in contrast to the surrounding scaffold and were consequently not detectable using conventional greyscale threshold methods. Therefore, using an image-based three-dimensional segmentation tool on the tomographic data revealed a multitude of non-stained cells. In addition, the SR-microCT data were compared with data obtained from scanning electron microscopy, energy dispersive X-ray spectroscopy and histology, while further linking the initial cell density measured via a MTT assay to the pore size as determined by SR-microCT.

Effect of different ascorbate supplementations on in vitro cartilage formation in porcine high-density pellet cultures.

Articular cartilage has only very limited potential for self-repair and regeneration. For this reason, various tissue engineering approaches have been developed to generate cartilage tissue in vitro. Usually, most strategies require ascorbate supplementation to promote matrix formation by isolated chondrocytes. In this study, we evaluate and compare the effect of different ascorbate forms and concentrations on in vitro cartilage formation in porcine chondrocyte high-density pellet cultures. l-ascorbate, sodium l-ascorbate, and l-ascorbate-2-phosphate were administered in 100 microM, 200 microM, and 400 microM in the culture medium over 16 days. Pellet thickness increased independently from the supplemented ascorbate form and concentration. Hydroxyproline content increased as well, but here, medium concentration of AsAP and low concentration of AsA showed a more pronounced effect. Proteoglycan and collagen formation were evaluated histologically and could be proven in all supplemented cultures. Non-supplemented cultures, however, showed no stable matrix formation at all. Effects on the gene expression pattern of cartilage marker genes (type I and type II collagen, aggrecan, and cartilage oligomeric matrix protein (COMP)) were studied by real-time RT-PCR and compared to non-supplemented control cultures. Expression level of cartilage marker genes was elevated in all cultures showing that dedifferentiation of chondrocytes could be prevented. Again, all supplementations caused a similar effect except for low concentration of AsA, which resulted in an even higher expression level of all marker genes. Besides that, we could not detect a pronounced difference between ascorbate and its derivates as well as between the different concentrations.