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. 2015 May 1;4(1):266–277. doi: 10.1089/biores.2015.0004

FIG. 7.

FIG. 7.

(a–c) Histological evaluation demonstrated positive markers for bone differentiation in long-term cultures. Deposits for glycosaminoglycans (Alcian blue staining), sparsely calcium deposits (Alizarin red staining), and several mineral deposits (von Kossa staining) were observed mainly toward the rand layer in 4-week static cultures. (a) The rand layer of a 4-week statically cultured collagen scaffold shows blue-stained glycosaminoglycan deposits (black arrowhead) (Alcian blue stain). (b) Red-stained calcium deposits (black arrows) are found in the rand layer of a statically cultured collagen scaffold at 4 weeks (Alizarin red staining). (c) Newly formed extracellular matrix with mineral deposits (black arrows) is stained with von Kossa staining in the rand layer of a statically cultured collagen scaffold at 4 weeks. (d–f) In the centrum of statically cultured scaffolds, less signs of bone differentiation markers are observed at 4 weeks, as shown in (d–f). (d) The fibrin clot at the centrum of a statically cultured scaffold (arrowheads) is bordered on very small glycosaminoglycan deposits (black arrows) inside the collagen scaffold (Alcian blue staining). (e) Alizarin red staining shows no signs for red calcium deposits in the collagen scaffold around the centrally placed fibrin clot (arrowheads) (Alizarin red staining). (f) The centrally placed fibrin clot shows a brownish ground staining with von Kossa (arrowheads), while there are only very few dark, stained mineralized deposits (black arrow) in between the surrounding collagen matrix in statically cultured scaffolds (von Kossa staining). (g–i) Histology of dynamically cultured scaffolds in bioreactor (von Kossa staining). (g) The section of a 4-week dynamically cultured scaffold shows mineral deposits centrally and peripherally (black arrows) and disintegration of the collagen matrix (white arrows). The edge of the remaining scaffold is marked by black arrowheads. (h) A higher magnification of a dynamically cultured scaffold is shown with a centrally located mineralized matrix (black arrows) and disintegration of the collagen matrix (white arrows) (von Kossa staining). (i) At 4 weeks of culture, part of the fibrin (black arrowheads) with some embedded cells (black arrow) is still seen centrally in the dynamically cultured scaffold. Adjacent regions with new extracellular matrix formation (white arrows) can be observed.