*2.3. Influence of TGF-β on Osteogenic Differentiation*

We tested the influence of TGF-β3 on the osteogenic differentiation of hMSC, seeded in monophasic scaffolds from mineralized salmon collagen. Specific ALP activity after 14 days of osteogenic stimulation showed a significant (*p* < 0.05) decrease in the presence of TGF-β3 both for hMSC and osteogenically prestimulated hMSC (Figure 4). The experiment was also performed to test the effectivity of osteogenic pre-stimulation in the monolayer, and a significantly (*p* < 0.001) higher specific ALP activity was detected both at day 1 and day 14.

**Figure 4.** Specific ALP activity of hMSC which were cultivated in monophasic scaffolds from mineralized salmon collagen under osteogenic stimulation (10−<sup>7</sup> M Dex, 10 mM β-GP, 12.5 μg/mL AAP = osteogenic supplements, OS+) in the presence and absence of 10 ng/mL TGF-β3. Dark bars: cells were prestimulated with OS+ in monolayer culture for 9 days before seeding the scaffolds. Cells of two different donors were used, each *n* = 3 per condition. ALP activity was related to cell number. Values are presented as mean (*n* = 6) +/− standard deviation. Significances from 2-way ANOVA were as follows: d1 ↔ d14 *p* < 0.001; d1 ↔ d14TGFβ *p* < 0.05; d14 ↔ d14TGFβ *p* < 0.05; non-differentiated ↔ pre-differentiated *p* < 0.001.
