4.1. Experimental Design at a Glance

Human mesenchymal stem cells were harvested from the femoral bone cavity of healthy donors (n = 6). A total of 2.5 × 10⁵ cells of each donor were seeded into T-150 flat bottom flasks and cultivated in expansion medium containing DMEM LG, 10% FCS, and 1% Penicillin/Streptomycin. After 5 days of expansion, cells from each donor were transferred into eight 35 mm diameter Petri dishes. Dish 1 was treated as a negative control (CTRL) and subsequently received DMEM low glucose 10% FCS, and 1% Penicillin/Streptomycin as a media. Dishes 2–8 were differentiated towards the osteogenic lineage using the standard osteogenic supplements dexamethasone (end concentration 100 nM), ascorbic acid (end concentration 50 µM), and ß-glycerol phosphate (end concentration 10 nM) using DMEM low glucose with 10% FCS but no Penicillin/Streptomycin as basal cell culture media. Each of the dishes 3–8 received a specific dose of an anti-infective agent: 8 µg/mL Gentamicin (Dish 3), 60 µg/mL Gentamicin (Dish 4), 20 µg/mL Vancomycin (Dish 5), 150 µg/mL Vancomycin (Dish 6), 2 µg/mL Voriconazole (Dish 7), and 5 µg/mL Voriconazole (Dish 8).

1. Group CTRL: Cell culture with DMEM low glucose containing 10% FCS and 1% Penicillin/Streptomycin.

2. Group OM: Osteogenic differentiation with DMEM low glucose containing 10% FCS and osteogenic supplements (100 nM dexamethasone, 50 µM ascorbic acid, 10 nM ß-glycerol phosphate).

3. Group GEN1: Osteogenic differentiation with DMEM low glucose containing 10% FCS and osteogenic supplements (100 nM dexamethasone, 50 µM ascorbic acid, 10 nM ß-glycerol phosphate), and Gentamicin (8 µg/mL).

4. Group GEN2: Osteogenic differentiation with DMEM low glucose containing 10% FCS and osteogenic supplements (100 nM dexamethasone, 50 µM ascorbic acid, 10 nM ß-glycerol phosphate), and Gentamicin (60 µg/mL).

5. Group VAN1: Osteogenic differentiation with DMEM low glucose containing 10% FCS and osteogenic supplements (100 nM dexamethasone, 50 µM ascorbic acid, 10 nM ß-glycerol phosphate), and Vancomycin (20 µg/mL).

6. Group VAN2: Osteogenic differentiation with DMEM low glucose containing 10% FCS and osteogenic supplements (100 nM dexamethasone, 50 µM ascorbic acid, 10 nM ß-glycerol phosphate), and Vancomycin (150 µg/mL).

7. Group VOR1: Osteogenic differentiation with DMEM low glucose containing 10% FCS and osteogenic supplements (100 nM dexamethasone, 50 µM ascorbic acid, 10 nM ß-glycerol phosphate), and Voriconazole (2 µg/mL).

8. Group VOR2: Osteogenic differentiation with DMEM low glucose containing 10% FCS and osteogenic supplements (100 nM dexamethasone, 50 µM ascorbic acid, 10 nM ß-glycerol phosphate), and Voriconazole (5 µg/mL).

Media change was every two days. After 21 days, the cell culture was terminated, and the mineral content was accessed. Samples for the analyses of the supernatant were collected on days 7, 14, and 21.