Fig 1.
After the wetted foam pack was kneaded according to the manufacturer’s recommendations, it was pressed into one well of a 6-well plate using a lid of a 50 ml tube to achieve equal distribution of the foam in the well (a). A 5 mm biopsy punch (b, Δ) was used to obtain cylindrically shaped scaffolds (b, *) from the compressed foam (b,→).
Table 1.
Overview regarding the used assays and observation time points.
Table 2.
Primers used for qPCR analyses.
Fig 2.
Definition of the cell population as MSC.
The cell populations were able to differentiate into chondrogenic lineage as demonstrated by the orange-colored extracellular chondrogenic matrix in Safranin O/Fast Green staining (a) and in adipogenic lineage, as shown by red-colored vacuoles in Oilred-O staining (b). Furthermore, the cells exhibit the MSC-specific surface characteristics (c): (i) corresponds to CD90, (ii) to CD105, (iii) to CD73 and (iv) to the negative control composed of CD14, CD20, CD34 and CD45. The gray bar in the left part of each graph shows the results of the isotype control being labeled with murine antibodies whilst the black bar on the right shows the results of human MSC being labeled with human antibodies. The x-axis shows the fluorescence intensity in logarithmic scale, the y-axis represents a height-normalized linear scale of cell number. Results are shown representatively for one donor.
Fig 3.
3D-mCT reconstruction and 2D-mCT slice of one representative Vitoss (a, b) and Vitoss BA scaffold (c, d). Representative fluorescence staining of a Vitoss scaffold after 21 (e) and 42 days (f) of incubation, respectively a Vitoss BA scaffold after 21 (g) and 42 days (h). The distribution of BG particles within the 2D scaffold structure (d, →) can be anticipated by scrolling through a stack of 2D-images: poreless, high-density particles with a size of 90–150 μm can be identified as BG. Both scaffold types consist of porous β-TCP granules (b and d, Δ) and a collagen matrix (b and d, +). Scale bars refer to 500 μm. In fluorescence staining, the green-stained intact cells dominate in both groups. Several BG particles remained intact at D21 (g, *) and some cells grew with contact to the particles (g and h, →), partially forming clusters (h, Δ). On D42 the fluorescence intensity of the BG particles decreased (h, *) compared to intensity at D21. Magnification: 40-fold.
Fig 4.
PrestoBlue reduction as correlate of cell vitality (a), cell proliferation measured via dsDNA content (b) and pH alteration (c) during the incubation time (D1-D42). Values are shown as means with standard error of the mean. (*) indicates significant differences.
Fig 5.
Correlates of osteogenic differentiation.
ALP activity (a) and quantitative gene expression of RUNX2 (b), SPP1 (c), and COL1A (d) during the incubation time (D1-D42). Since ALP activity was significantly higher at any observation point for D7, D14, D21, and D42 when compared to D1 in both groups, significances are not indicated in the figure for reasons of clarity. Gene expression was normalized to gene expression on D1 and is therefore shown as x-fold expression. Values are shown as means with standard error of the mean. (*) indicates significant differences.