*4.3. Osteogenic Differentiation of hMSC in Mineralized Salmon Collagen Scaffolds*

Monophasic scaffolds from mineralized salmon collagen (d = 6 mm, h = 3 mm) were prepared as already published [7] and sterilized by γ-irradiation before use in cell culture. Scaffolds were seeded with 2 × 104, 5 × <sup>10</sup>4, and 1 × <sup>10</sup><sup>5</sup> cells in 50 <sup>μ</sup>L of expansion medium. After 30 min of initial adhesion, further 500 μL of expansion medium were added to each scaffold. Cell-seeded scaffolds were cultivated for up to 4 weeks, with medium change every 3–4 days. Osteogenic differentiation medium contained Minimal essential medium α-modification (α-MEM) (Biochrom Berlin, Germany), supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (all from Biochrom), 10−<sup>7</sup> M Dex, 12.5 μg/mL AAP and 10 mM β-GP (all from Sigma, Taufkirchen, Germany). Since we wanted to analyze the effect of TGF-β on the osteogenic differentiation additionally 10 ng/mL TGF-β3 (Miltenyi, Bergisch Gladbach, Germany) were added to one experimental group. After 1, 14 and 28 days samples for DNA and ALP quantification were washed twice and frozen at −80 ◦C.

#### *4.4. Cultivation of Osteochondral Constructs*

Biphasic scaffolds from jellyfish collagen and mineralized salmon collagen (d = 6 mm, h = 8 mm) were sterilized by γ-irradiation before use in cell culture. Sodium alginate (Sigma) was dissolved in Ca2+ free DMEM (Sigma) without any further supplements at 12 mg/mL and the solution was filtered through a syringe filter with 0.45 <sup>μ</sup>m pore size. 5 × 105 cells were suspended in 50 <sup>μ</sup>L of alginate solution and the suspension was applied to the top of each biphasic scaffold. With 6 mm diameter and 3 mm height the chondral phase of the biphasic scaffold has an approximate volume of 85 mm<sup>3</sup> resulting in a seeding density of 6 × <sup>10</sup><sup>6</sup> cells/cm3. After 15 min of incubation at 37 ◦C, 1 mL of sterile CaCl2 (100 mM) solution was added and the constructs were incubated for further 15 min for the formation of alginate gel. The constructs were washed with expansion medium and cultivated further with chondrogenic differentiation medium (DMEM high glucose (Gibco, Dublin, Ireland, distributed by Thermo Fisher, Waltham, MA, USA), 100 U/mL penicillin, 100 μg/mL streptomycin, 1% ITS-X-Mix (Gibco; results in concentrations of 10 μg/mL insulin, 5.5 μg/mL transferrin, 6.7 ng/mL sodium selenite and 2 μg/mL ethanolamine in the medium), 0.35 μM proline, 50 μg/mL AAP, 10−<sup>7</sup> M Dex, 0.15% bovine serum albumin (BSA) (all Sigma) and 10 ng/mL TGF-β3 (Miltenyi)) for 9–12 days with medium changes every 3–4 days. At the same time, hMSC from the same batch were cultivated in T-flasks with α-MEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin, 10−<sup>7</sup> M Dex, 12.5 μg/mL AAP and 10 mM βGP. After 9 to 12 days of separate cultivation, biphasic scaffolds seeded with chondrogenically pre-differentiated hMSC were gently dried on sterile filter paper, flipped, that the mineralized layer was on top of the scaffold and

were seeded in the mineralized salmon collagen layer with 5 × <sup>10</sup><sup>4</sup> osteogenically pre-differentiated hMSC in 50 <sup>μ</sup>L cell culture medium, resulting in a seeding density of 6 × 105 cells/cm3. The constructs were cultivated up to 21 days of total cultivation time with osteochondral medium consisting of DMEM high glucose supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 1% ITS-X-Mix, 5 ng/mL TGF-β3, 10−<sup>7</sup> M Dex, 0.35 μM proline, 0.15% BSA and 50 μg/mL AAP. For each time point of the experiment 9 biphasic scaffolds were used (3 for gene expression analysis, 3 for measurement of collagenII and sGAG, 1 for MTT staining, 1 for fluorescence staining and 1 for histological staining).
