Antibiotic antimycotic mixture

Human tendon stem cells (hTSCs) were isolated from supraspinatus tendon specimens collected during arthroscopic rotator cuff repair, as previously reported [4 (link)]. The isolated hTSCs were cultured in minimal essential medium alpha modification (α-MEM) (Merck) supplemented with 2 mM L-glutamine (Euroclone), 1% antibiotic-antimycotic mixture (Euroclone), and 20% (v/v) fetal bovine serum (FBS) (HyClone, Thermo Fisher Scientific) at 37°C in a 5% CO₂ and 95% air-humidified atmosphere. The medium was changed every 2-3 days.

Human tendon stem cells (hTSCs) were isolated from supraspinatus tendon specimens collected during arthroscopic rotator cuff repair, according to a previously reported procedure [15 (link)]. The protocol study was approved by the Hospital Ethical Committee with authorization number 2642 (Sept. 19, 2011). Samples from supraspinatus tendons (4–8 mm wide) were collected from 6 patients after signed informed consent, kept in HypoThermosol (BioLife Solutions) at 4°C, and processed separately within 24 h, according to the procedure described below. Samples were washed with phosphate-buffered saline (PBS) (Euroclone), cut into small pieces, and digested for 90 min with collagenase type I (3 mg/mL; Worthington) and dispase (4 mg/mL; Gibco, Life Technologies) in PBS at 37°C. After centrifugation, cell pellets were resuspended in the following culture medium: α-Minimal Essential Medium (α-MEM) (Sigma-Aldrich) supplemented with 2 mM glutamine (Euroclone), 1% antibiotic-antimycotic mixture (Euroclone), and 20% (v/v) fetal bovine serum (FBS) (HyClone, Thermo Fisher Scientific). Cells were then filtered with a cell strainer (70 mM; BD Falcon) and plated in 150 cm² dishes. Adherent cells were cultured at 37°C in a humidified atmosphere of 5% CO₂. The medium was changed every 2-3 days. All experiments were carried out with cells at passage four to six after isolation [17 (link)].

To assess the effects of the Lipogems product on the osteogenic differentiation capacity of hTSCs, cells were plated at a concentration of 3 × 10⁴ cells/cm² and preconditioned with the Lipogems product for 96 h in normal growth medium and then switched to the osteogenesis induction medium, which was constituted of DMEM-low glucose (Sigma-Aldrich), 10% FBS (HyClone, Thermo Fisher Scientific), 4 mM L-glutamine (Euroclone), and 1% antibiotic-antimycotic mixture (Euroclone), supplemented with 0.1 μM dexamethasone, 50 μg/mL L-ascorbic acid-2-phosphate, and 10 mM β-glycerophosphate (all reagents from Sigma-Aldrich) for 17 days. At day 17, Alizarin Red solution (Millipore) was used to detect calcium deposition in derived osteoblasts according to the manufacturer's instruction. All photomicrographs were acquired with Axiovert 40 microscope (Zeiss) equipped with a Moticam 2300 camera (Motic). The osteogenic medium was changed every 2-3 days.

Human tendon stem cells (hTSCs) were isolated from supraspinatus tendon specimens collected during arthroscopic rotator cuff repair, according to our previous procedure [22 (link)]. The isolated hTSCs were cultured in normal growth medium composed of α-Minimal Essential Medium (α-MEM) (Sigma-Aldrich) supplemented with 2 mM glutamine (Euroclone), 1% antibiotic-antimycotic mixture (Euroclone), and 20% (v/v) fetal bovine serum (FBS) (HyClone, Thermo Fisher Scientific) at 37°C in a humidified atmosphere of 5% CO₂. The medium was changed every 2-3 days. All experiments were carried out with cells at passage four after isolation.

Cells were plated at the concentration of 3 × 10⁴ cells/cm² and cultured in the osteogenesis induction medium, which was constituted of DMEM-low glucose (Sigma-Aldrich), 10% FBS (HyClone, Thermo Fisher Scientific), 4 mM L-glutamine (Euroclone), and 1% antibiotic-antimycotic mixture (Euroclone), supplemented with 0.1 μM dexamethasone, 50 μg/mL L-ascorbic acid-2-phosphate, and 10 mM β-glycerophosphate (all reagents from Sigma-Aldrich) for 17 days. The osteogenic medium was changed every other day. At day 17, Alizarin Red solution (Millipore) was used to detect calcium deposition in derived osteoblasts, according to the manufacturer's instruction. All photomicrographs were acquired with an Axiovert 40 microscope (Zeiss) equipped with a Moticam 2300 camera (Motic). The expression of osteogenic marker ALP was determined by real-time PCR on days 5 and 17, as described above [17 (link)].

hTSCs were plated at a concentration of 3 × 10⁴ cells/cm² in normal growth medium, and then switched to the osteogenesis induction medium, which was constituted of DMEM-low glucose (Sigma-Aldrich), 10 % FBS (HyClone, Thermo-Fisher Scientific), 4 mM L-glutamine (Euroclone), 1 % antibiotic-antimycotic mixture (Euroclone), supplemented with 0.1 μM dexamethasone, 50 μg/ml L-ascorbic acid-2-phosphate, and 10 mM β-glycerophosphate (all reagents from Sigma-Aldrich) for 17 days. At day 17, Alizarin Red solution (Millipore) was used to detect calcium deposition in derived osteoblasts, according to the manufacturer’s instruction. All photomicrographs were acquired with an Axiovert 40 microscope (Zeiss) equipped with a Moticam 2300 camera (Motic). The osteogenic medium was changed every 2–3 days.

To assess the effects of the Lipogems product on the adipogenic differentiation capacity of hTSCs, cells were plated at a concentration of 3 × 10⁴ cells/cm² and preconditioned with the Lipogems product for 96 h in normal growth medium and then switched to DMEM-low glucose (Sigma-Aldrich), 10% FBS (HyClone, Thermo Fisher Scientific), 4 mM L-glutamine (Euroclone), and 1% antibiotic-antimycotic mixture (Euroclone), with the addition of the mesenchymal stem cell adipogenesis kit (Millipore) for 21 days, according to the manufacturer's instructions. At day 21, Oil Red O solution (Millipore) was used to stain lipid droplets of derived adipocytes, according to the manufacturer's procedures. All photomicrographs were acquired with Axiovert 40 microscope (Zeiss) equipped with a Moticam 2300 camera (Motic). The adipogenic medium was changed every 2-3 days.