Cryoprobe in gelatin
After freezing was initiated with the maximum nitrogen flow rate, a spindle-shaped ice formation began to form, radiating outwards from the freezing zone, progressively becoming more spherical. Analogous to these observations, it was shown that the vertical freezing capacity of the probe was greatest in the middle part of the cold zone (Fig. 2). Even after 8 minutes, a temperature of -51 ± 2.9°C was measured on sensor C (Fig. 2) while an average temperature of -22.5 ± 4.1°C was read at the distal and the proximal ends of the cold zone (sensors A and D, Fig. 2). Even after 15 minutes, at the end of the freeze, the temperature in the centre of the cold zone was lower with -48.6 ± 4.6°C (sensor B, Fig. 2) and -61.5 ± 1.5°C (sensor C, Fig. 2). On the other hand, temperatures at the end of the cold zone were measured at -34 ± 3.7°C (sensor B, Fig. 2) and -36 ± 4.5°C (sensor A, Fig. 2).
The time- and place-dependent decrease in temperature in the horizontal direction is shown as a system of curves, with the distance of the sensors from the tip of the probe as the variable (Fig. 3). The temperature drop at the beginning of freezing near the probe (5 mm, sensor A, Fig. 3) was very steep, with a maximum of 49 ± 18.0°C per minute between the first and the second, as well as the second and the third minute after beginning of freezing. More than 90% (-106°C) of maximum cooling (-113°C) was achieved after only 7 minutes. With increasing distance from the probe and duration of freezing, the cooling rate decreased markedly (temperature decrease per minute); e.g., a distance of 10 mm from the probe required 8 minutes after beginning of freezing to reach a temperature of -50°C. A maximum cooling rate of 27°C/minute was reached between the second and the third minute after beginning of freezing. The temperature drop at a distance of 20 mm from the probe (sensor D, Fig. 3) is nearly linear, with a maximum value of 7 ± 1.77°C/ minute.
Two cryoprobes in gelatin
To the eye, there was no difference in freezing when a single probe was employed compared to when two cryoprobes were used simultaneously. As the edge of the cold zone advanced, there was an initial melting of the ice crystals at the equator of both edges (Fig. 4). Later on, the crystals merged into a confluent sphere. After 15 minutes, at the end of freezing, the ice sphere was 7 ± 0.9 cm wide and 6.4 ± 0.7 cm high. Fig. 5 shows the change in temperature as a function of the freezing time between two active cryoprobes (sensor A, Fig. 5), as well as the synchronous temperature measurements next to both probes (sensors B and C, Fig. 5). A synergistic freezing effect was seen between the probes, with a higher cooling rate of up to -66°C between the fourth and the fifth minute of freezing, as well as a much lower minimum temperature of -117°C, compared to the measurements in the same intervals when using only one probe (≤ -60°C).
In vivo freezing
The decrease in temperature was less pronounced when freezing living bone with one cryoprobe than for the in vitro measurements (Fig. 6). The ice front also did not spread as far. Analogous to the in vitro trials, the highest rate of cooling was measured near the probe (5 mm, sensor A, Fig. 6), where the temperature sank to -77 ± 3°C. Even at a distance of 10 mm from the probe, a temperature of -41 ± 2°C could be reached. Accordingly, as the distance from the probe increased, the temperature drop also was decreased.
The measurements with two cryoprobes show the synergistic freezing effects through the confluence of two freezing zones, which already was observed in the in vitro trials. The minimum temperatures reached between the probes, on the other hand, were higher than in vitro because of the body heat of the animals. Nevertheless, with a temperature of -65° ± 3°C (sensor A, Fig. 7), this was lower than the corresponding measurement points B and C (Fig. 7), which were 1 cm away from only one probe. The temperatures reached here (-39 ± 4°C) corresponded approximately to the temperatures measured for one probe at the same intervals. No systemic or local intraoperative complications were observed.
Macroscopically, the cryosurgical-treated bone section showed a circular necrosis which was sharply bounded and distinct from neighbouring bone and marrow tissue. Histological examination showed an extension of the bone necrosis corresponding to the -10°C isotherm (Fig. 8), with a continuous transition from full necrosis to vital bone (Fig. 9). The marrow necrosis is more homogeneous, extends much farther and passes the region of compact bone necrosis by about 2 cm.
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