*2.2. Evaluation of Optimal Seeding Density for the Osteogenic Differentiation of hMSC*

Single scaffolds from mineralized salmon collagen were seeded with hMSC at three different densities, 2.4 × 105 cells/cm3, 6 × 105 cells/cm3, and 1.2 × 106 cells/cm<sup>3</sup> and cultivated under osteogenic stimulation for 14 and 28 days. Number of viable cells, visualized by MTT staining (Figure 3a) was still highest in the scaffolds with the highest initial seeding density after 28 days of osteogenic stimulation. Quantitative analysis of cell number (Figure 3b) confirmed these findings. Furthermore, it was shown, that cell number did not increase between d14 and d28 of cultivation. Osteogenic differentiation was evaluated by quantification of specific alkaline phosphatase (ALP) activity (Figure 3c). Highest specific ALP activities were obtained for the constructs with the lowest seeding density; however, due to high variations between single samples the effect was not statistically

significant. Furthermore, the ALP activity in scaffolds seeded with the lowest cell density was close to the detection limit of the colorimetric test. Therefore, for the seeding of biphasic scaffolds, the intermediate cell density (6 × <sup>10</sup><sup>5</sup> cells/cm3) was chosen, since specific ALP activity was still higher compared to the highest seeding density.

**Figure 1.** Biphasic scaffolds from mineralized salmon collagen (**lower layer**) and fibrillized jellyfish collagen (**upper layer**) in dry state (**a**) as well in wet state (**b**). Arrows indicate the transition zone between the layers. Scale bar represents 5 mm.

**Figure 2.** SEM image of a cross section of a biphasic scaffold at the transition zone between jellyfish collagen (**upper layer**) and mineralized salmon collagen (**lower layer**). Scale bar represents 200 μm. Insert showing the transition zone with higher magnification; scale bar represents here 20 μm.

**Figure 3.** (**a**) MTT staining of osteogenically differentiated hMSC seeded in mineralized salmon collagen scaffolds at different densities and cultivated for 28 days; (**b**) cell number, calculated from DNA content and (**c**) specific ALP activity (ALP activity related to cell number) of osteogenically differentiated hMSC in salmon collagen scaffolds with different seeding densities after 14 and 28 days of cultivation, *n* = 3, mean +/− standard deviation. Significances from 2-way ANOVA were as follows: Cell number: d14 <sup>↔</sup> d28 n.s.; 2.4 <sup>×</sup> <sup>10</sup><sup>5</sup> <sup>↔</sup> <sup>6</sup> <sup>×</sup> <sup>10</sup><sup>5</sup> n.s., 2.4 <sup>×</sup> <sup>10</sup><sup>5</sup> <sup>↔</sup> 1.2 <sup>×</sup> <sup>10</sup><sup>6</sup> *<sup>p</sup>* < 0.05; 6 <sup>×</sup> <sup>10</sup><sup>5</sup> <sup>↔</sup> 1.2 <sup>×</sup> 106 *<sup>p</sup>* < 0.05, specific ALP activity: d14 <sup>↔</sup> d28 n.s.; 2.4 <sup>×</sup> \* 10<sup>5</sup> <sup>↔</sup> <sup>6</sup> <sup>×</sup> \* 10<sup>5</sup> n.s., 2.4 <sup>×</sup> 105 <sup>↔</sup> 1.2 <sup>×</sup> 106 *<sup>p</sup>* < 0.05; 6 <sup>×</sup> <sup>10</sup><sup>5</sup> <sup>↔</sup> 1.2 <sup>×</sup> 106 n.s.
