VITRIFICATION OF ARTICULAR-CARTILAGE BY HIGH-PRESSURE FREEZING

For more than 20 years, high-pressure freezing has been used to cryofi x bulk biological specimens and reports are available in which the pot ential and limits of this method have been evaluated mostly based on m orphological criteria. By evaluating the presence or absence of segreg ation patterns, it was postulated that biological samples of up to 600 mu m in thickness could be vitrified by high-pressure freezing. The c ooling rates necessary to achieve this result under high-pressure cond itions were estimated to be of the order of several hundred degrees ke lvin per second. Recent results suggest that the thickness of biologic al samples which can be vitrified may be much less than previously bel ieved. It was the aim of this study to explore the potential and limit s of high-pressure freezing using theoretical and experimental methods . A new high-pressure freezing apparatus (Leica EM HPF), which can gen erate higher cooling rates at the sample surface than previously possi ble, was used. Using bovine articular cartilage as a model tissue syst em, we were able to vitrify 150-mu m-thick tissue samples. Vitrificati on was proven by subjecting frozen-hydrated cryosections to electron d iffraction analysis and was found to be dependent on the proteoglycan concentration and water content of the cartilage. Only the lower radic al zone (with a high proteoglycan concentration and a low water conten t compared to the other zones) could be fully vitrified. Our theoretic al calculations indicated that applied surface cooling rates in excess of 5000 K/s can be propagated into specimen centres only if samples a re relatively thin (<200 mu m). These calculations, taken together wit h our zone-dependent attainment of vitrification in 150-mu m-thick car tilage samples, suggest that the critical cooling rates necessary to a chieve vitrification of biological samples under high-pressure freezin g conditions are significantly higher (1000-100000 K/s) than previousl y proposed, but are reduced by about a factor of 100 when compared to cooling rates necessary to vitrify biological samples at ambient press ure.