Processing and sterilization of the procured bone

The study was approved by the Institutional Ethics Board and informed consents were obtained from the donors. Femoral heads were harvested intraoperatively from patients (age range 53–68 years) undergoing total hip arthroplasty. The procured bone material was stored at −80°C in sterile polyethylene containers. Later, batches of collected femoral heads were processed in a biological safety cabinet MSC-9 (Thermo Fisher Scientific, USA). After thawing for 8 h at room temperature and removal of extraneous tissues, the heads were cut with a medical hand saw into blocks (cubes) with the average side length of 0.5 cm. The blocks were incubated overnight at 4°C in 400 ml saline containing 1.0 g of ciprofloxacin. After that, the blocks were processed and sterilized with three different methods.

Chemical sterilization protocol included washing of the blocks in 3.0% sodium hydrocarbonate solution with a brush and soaking in saline for 30 min. After drying at room temperature, bone blocks were packed in polyethylene bags filled with a disinfectant solution and stored at −20°C. The solution contained a mixture of citric acid, glucose, sodium bromide, 95% ethanol, nitrofural, dimethyl sulfoxide (DMSO), amikacin in distilled water (Patent RU2235462).

Thermal sterilization involved heating of the bone blocks placed into a sealed container with saline in Lobator SD-2 sterilizer (TELOS, Germany). The duration of the protocol was 94 min, while sterilization temperature (82.5°C) was sustained for at least 15 min. After cooling, saline was discarded via a safety valve and the container was stored at −20°C. This sterilization procedure is described in more detail elsewhere [24].

Processing of the bone blocks by washing with γ-irradiation was based on a previously published protocol and included our modifications [25]. Briefly, bone blocks were agitated in distilled water for 60 min at 59°C and cleaned in hydrodynamic water flow for 15 min. After that, three consecutive wash cycles were performed.

The first wash cycle consisted of 20 min in 10% sodium hydrocarbonate, 15 min in hydrodynamic flow, 20 min of sonication (45 Hz) in 10% sodium hydrocarbonate at 59°C, 15 min in hydrodynamic flow, 15 min in 10% sodium hydrocarbonate with agitation at 220 rpm, 15 min centrifugation at 1850 g.

The second wash cycle consisted of the same steps as previous only with distilled water instead of sodium hydrocarbonate. A total of five repeats were performed.

The third cycle included washing in 3% hydrogen peroxide at 59°C in a shaking water bath (22), sonicator (45 Hz) and orbital shaker (220 rpm) for 20 min each. After that, the same wash sequence was completed with 70% ethanol.

Finally, bone blocks were agitated three times for 15 min in sterile distilled water at 59°C. The blocks were dried for 2 h at 45°C and quick-frozen at −80°C. Later, the blocks were lyophilized on HETO PowerDry PL3000 (Thermo Fisher Scientific, USA) for 40 h, packed in double polyethylene bags (Clinipack, Russia) with the average length of 5 cm and γ-irradiated on a conveyor belt with a dose of 25 kGy at a speed of 150 cm/min. The resulting average exposition time was 2 s. Packed sterile blocks were stored at −20°C.