Collagenase 2

Fat tissues (obtained from hip or abdomen) were cut into mm-size pieces using a scalpel. The samples were washed several times with phosphate buffered saline (PBS). To release Ad-MSCs from the tissue samples were incubated in a 0.7% collagenase II solution (Biochrom, Berlin, Germany) for approximately 30 minutes at 37 °C. The addition of culture medium (DMEM 4.5 g/L glucose, 10% FCS, 100 U/mL penicillin, 100 µg/mL Streptomycin) stopped the collagenase II activity. After centrifugation at 600 g for 10 min the Ad-MSC pellet was re-suspended in culture medium for expansion (37 °C, 5% CO₂, humidified atmosphere). Experiments were performed in passage 3 or 4 when Ad-MSCs were negative (determined by RT-PCR) for CD14 and positive for CD73, CD90, and CD105, as reported earlier [21 (link),39 (link)].

Adult Wistar rats (Charles River) were euthanized in CO₂ anesthesia by cervical dislocation. Organ harvest was approved by the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES: AZ 10/13). For one series of ESM skeletal muscle from both hind limbs (total 20‐30 g) of one Wistar rat (6‐8 weeks old) was collected in basal medium (DMEM/F12, 5% fetal bovine serum [FBS], 2 mmol/L glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin) on ice. Connective tissue and tendons were removed and the muscle was minced to a fine slurry. This was digested for 90 min at 37°C using 2.5 mg/ml Collagenase II (Biochrom) and 20 µg/ml DNAse I (Calbiochem) in basal medium. The tissue suspension was subsequently passed through filters with gradually decreasing mesh size (250 100 40 µm, BD Biosciences). The cell suspension was then preplated for 1 hour in basal medium on cell culture dishes to reduce the number of fibroblasts. The non‐attached cells were resuspended in proliferation medium (basal medium supplemented with 10 ng/ml bFGF; PeproTech), 10 ng/ml EGF (PeproTech), Insulin‐Transferrin‐Selenite (ITS) + X (100x; Invitrogen) and expanded for 5 days on uncoated flasks without further passaging. For implantation experiments transgenic Lewis rats with ubiquitous expression of EGFP were used for skeletal myocyte preparation.28

Cell-seeded collagen gels (six 0.5 mL-gels per experimental group) were removed from the transwell inserts and incubated with collagenase II solution (3 mg/mL collagenase II, 235 U/mg, Biochrom, Berlin, Germany in α-MEM, 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin, 3 mM CaCl₂) for 1 h at 37 °C. The digests were transferred to 15 mL tubes, washed with PBS and centrifuged. The pellets were washed with PBS and RNA was isolated from these pellets as well as from the osteoblast-seeded transwell membranes using a commercially available kit (peqGOLD MicroSpin Total RNA Kit, Peqlab, Erlangen, Germany). cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, part of Thermo Fisher Scientific) according to manufacturer’s instructions.

Briefly, cancellous bone was disintegrated mechanically and washed 3–5 times with Dulbecco’s phosphate buffered saline (DPBS). After 1 h of collagenase digestion (DPBS, 0.07% collagenase II—Biochrom AG, Berlin, Germany) at 37 °C, cancellous bone pieces were washed with DPBS and released OBs were transferred to cell culture flasks in growth medium (MEM/Ham’s F12, 10% FCS, 2 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μM l-ascorbate-2-phosphate, 50 μM β-glycerol phosphate) for expansion. Medium was changed every 3–4 days [22 (link),23 (link)].

Periosteal cells were isolated from mastoid autografts (0.5 cm²) taken from four independent donors undergoing mastoidectomy according to a method previously described [7 ]. In brief, the periosteal flap was rinsed with Hanks solution (Biochrom, Berlin, Germany) three times, minced and digested for 3 hours in Dulbecco’s modified eagle medium (DMEM)/Ham’s F12 medium (Biochrom) containing 10,000 U/ml collagenase II (Biochrom), 10% human allogenic serum (German Red Cross, Berlin, Germany), 2.5% Hepes (Biochrom) and 1% penicillin/streptomycin solution (Biochrom). Subsequently, the cells were harvested, resuspended in DMEM/Ham’sF12 medium containing 10% human allogenic serum, plated in cell culture dishes (∅ = 15 cm), and allowed to attach for about 4–6 days.