The Erbokryo CS-6 System is a commercially available cryotherapy device which consists of a device with a control unit and vacuum-isolated flexible tubes leading to the cryoprobes. The case also contains a 47 liter capacity vacuum-isolated tank (supply Dewar) with liquid nitrogen which is heated electrically. The probes are started by the resulting vapor pressure of up to 15 bar. The liquid nitrogen (-196°C) reaches the 3 cm long freezing zone at the tip of the probe through a central delivery tube and then vaporizes through heat absorption. The flow of nitrogen can be chosen for each probe at four levels, from 25–100% up to a maximum flow rate of 1.5 l/min. The nitrogen gas then flows through a coaxial external pipe back into the tank. A total of 6 cryoprobes can be used simultaneously.
In the first part of the experiment, a transparent gelatin was used at room temperature and the formation of ice crystals around a single probe as well as two coupled probes photographed every two minutes. At the same time, the temperature gradients were determined horizontally and vertically within the cold zone every minute using the computer-guided Erbekryo CS-6 temperature couples (Thermoelement Typ K, galvanically separated channels, 6 separate BF type channels for external temperature measurement) which allow up to 6 temperature measurements simultaneously. These sensors, which have a diameter of 1.1 mm, were placed by eye every 0.5 cm at a distance of 2 cm from the centre of the probe using a special plexiglass holder. The vertical measurement was carried out in 1 cm intervals from the probe over the entire freeze zone. For the simultaneous use of two probes 2 cm apart, temperature distributions were determined half-way between the two probes and diametrically at 1 cm intervals around each probe. Four different measurements were carried out for each trial, each in gelatin with different probes (all by the same manufacturer).
In the second part of the experiment, a total of 4 freezes were carried out on both hind legs of a sheep under general anaesthesia. Holes 3.6 mm in diameter were drilled perpendicular to the shaft axis (Fig. 1a/1b). The cryoprobes were introduced and the remaining space filled with 10% NaCl solution as a coupling medium. The proximal tibial metaphysis was frozen using one cryoprobe (Fig. 1a) and then the distal diaphysis-metaphysis transition area of the femur was frozen using two probes as described for the in vitro freezings (1b). The thermocouples were introduced into the drilling holes (diameter 1.2 mm) at distances of 7.5 mm, 10 mm, 12.5 mm and 15 mm from the center of the cold area using one cryoprobe (Fig. 1a) and, using two probes simultaneously, at a distance of 10 mm (Fig. 1b). The exact position of the cryoprobes and the thermocouples was checked radiologically. Measurements were taken at one-minute intervals using a computer. Thermal influence of the measurement on the opposite side was avoided by alternating the freezings. Intraoperative control of blood pressure, heart rate, body temperature and acid-base balance (periodic blood gas analysis) were checked to detect potential complications.
One week postop the animal were sacrificed and the extent of necrosis determined histologically. The bone segments were decalcified (ethylene diamine tetraacetic acid), embedded in Technovit 7100 and sectioned for hematoxylin-and-eosin staining. The results of the trials and statistical evaluation were done in SPSS for Windows, Version 9.0.