When mixed cultures of mouse fibroblasts and hydroxyapatite (HA) granules were subjected to various stimuli, the fibroblasts could proliferate in a three-dimensional pattern around the HA granules. In this study, we investigated the effects of two physical stimuli: an electromagnetic field, which is thought to be effective for promotion of bone formation, and ultrasonic waves. Although mixed cultures of mouse fibroblasts and HA stimulated with an electromagnetic field of 1 gauss for 8 hrs or 16 hrs showed only 2-fold higher induction rates of threedimensionallike cell proliferation than that in the untreated control culture, stimulation for 24hrs resulted in 10-fold higher induction rates. Irradiation of cultures with ultrasonic waves of 5 W/cm² for 3 sec resulted in no induction of cell proliferation, but irradiation for 6 sec and for 9 sec resulted in 4-fold and 5-fold higher induction rates, respectively. The results indicate that the two types of physical stimulus tested in this study have optimal doses to induce the formation of a three-dimensional structure.

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Small preantral follicles would be a potentially rich source of genetic material, if they could be cryopreserved and then grown to maturity in vitro. The goal of this study was to develop a cryopreservation technique, which ensures high recovery and viability of bovine small preantral follicles after cryopreservation, for subsequent in vitro culture. The numbers of recovered and viable follicles were measured after each step of cryopreservation by the microscopic observation and dye exclusion (Hoechst 33258 and Trypan blue). Exposing the preantral follicles to 37°C or 24°C resulted in similar high viabilities (51.3% vs.46.8%), but the follicles cooled to 0°C exhibited a significant loss of viability (25.0%). Recovery rates (6.5% vs.22.2%)of vitrified preantral follicles in plastic French straws and cryotubes with nylon mesh filters for a bottom were quite low after vitrification procedures (equilibration, cryopreservation and thawing). Presumably, vitrified follicles tightly adhered to the walls of containers and micropipettes. In contrast, high recovery (76.3%) was obtained when follicles were embedded in collagen gels prior to the vitrification. Furthermore,30% of the collagen gel-embedded follicles were viable after the vitrification compared to 58.3% viability of non-vitrified follicles embedded in collagen gels. The recovery, viability and cell proliferation activity (incorporation of BrdU into the DNA of S-phase cells in non-vitrified and vitrified follicles embedded in collagen gels) were determined after 7 days in culture. The recovery rates and viability of the vitrified follicles were about half of the values obtained with non-vitrified (47.5% vs.91.7%; 18.4% vs.36.4%, respectively). Cell proliferation activity revealed that viable vitrified preantral follicles could grow in the same manner as viable non-vitrified follicles. In summary, bovine small preantral follicles embedded within collagen gels can be successfully cryopreserved with a high recovery rate by using the vitrification technique.

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