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Amelogenesis and mineralization in vitro
Cell Biology
International
Reports,
AMELOGENESIS
Vol. 5, No. 8, August
AND
MINERALIZATION
A.L.J.J.Bronckers, T.J.M.Bervoets Laboratory of Preventive Dentistry, De Boelelaan 1115, Amsterdam, There is a general lack of a reproducible culture system for studying the formation of enamel matrix and its subsequent mineralization in vitro (Slavkin, 1979) .A number of conditions for an optimal production of dentin and enamel matrix was investigated by biochemical and morphomctrical methods. Finest development and optimal matrix production and mineralization were found, when 2nd or 3rd day 2nd molars of hamsters were cultivated in upward position using the Trowel1 method in BGJb medium(pheno1 red and glutamine omitted, Flow), supplemented with 15%FBS (Gibco) , 250m/ml vit C (Bronckers, 1980), ZOO&/ml glutamine at pH = 7,3 at 37OC in a gasphase of 50%02+5%C02+45%N2. Medium was changed daily. At time of explantation of 2nd day 2nd molars no matrix was present (bell stage) . First predentin was. found in tile tip of the cusps after 3 days of culture. At day 7-8 the first dentin was observed. In contrast to the findings of Yamada et al. ( 1980) this dentin was van Kossa positive (calcium) Opposite to this dentin the first enamel matrix was found which showed a fine granular von Kossa staining. After 10 days of cultivation a thick layer of enamel
Fig. 1. Second day 2nd molar cultivated for 10 days. Toluidine blue, 1 pm epon section; a ameloblasts; em enamel matrix; p predentin: d dentin: o odontoblasts x 95 Received:
March
03OS-1651/8110807714J11$02.00/0
1981
771
1981
IN VITRO and J.H.M.WGltgens Vrije Universiteit ‘The Netherlands
matrix had been produced (Fig. 11, which was found to mineralize from the enamel dentin junction towards the ameloblasts (Fig. 2). In many of the cultured explants in the ameblasts in the bottom of the deep fissures an accumulation of secretory granules was found. Occasionally some local destruction was observed in these regions. apparently due to diffusion problems. All cultured explants (n = 85) were found to produce enamel matrix of the prismatic type in considerable amounts( 8-1099 of the total area). It can be concluded that for studying amelogenesis and mineralization, this culture system is a useful1 and reliable tool. References Slavkin, H C . ( 1979) Amelogenesis in vitro. Journal of Dental Research 58B, 735-741 Bronckers,A.L.J.J. and Wiiltgens,J.H.M(1980) Morphological and biochemical aspects of vit C deficiency during tooth germ development in vitro. Calcified Tissue International suppl 31, 13 Yamada,M., Bringas,P., Grodin,M .MacDougall, M and Slavkin, H . C . ( 1980) Developmental comparisons of murine secretory amelogenesis in viva, as xenografts on the chick chorio allantoic membrane and in vitro.Calcified Tissue International 31, 161-171
Fig 2. Detail left cusp. Von Kossa, lpm epon section: d dentin: em enamel matrix; a ameloblasts; si stratum intermedium; edj enamel dentin junction x 1025 Accepted:
June
0 1981 Academic
1981
Press Inc. (London)
Ltd.
International
Reports,
AMELOGENESIS
Vol. 5, No. 8, August
AND
MINERALIZATION
A.L.J.J.Bronckers, T.J.M.Bervoets Laboratory of Preventive Dentistry, De Boelelaan 1115, Amsterdam, There is a general lack of a reproducible culture system for studying the formation of enamel matrix and its subsequent mineralization in vitro (Slavkin, 1979) .A number of conditions for an optimal production of dentin and enamel matrix was investigated by biochemical and morphomctrical methods. Finest development and optimal matrix production and mineralization were found, when 2nd or 3rd day 2nd molars of hamsters were cultivated in upward position using the Trowel1 method in BGJb medium(pheno1 red and glutamine omitted, Flow), supplemented with 15%FBS (Gibco) , 250m/ml vit C (Bronckers, 1980), ZOO&/ml glutamine at pH = 7,3 at 37OC in a gasphase of 50%02+5%C02+45%N2. Medium was changed daily. At time of explantation of 2nd day 2nd molars no matrix was present (bell stage) . First predentin was. found in tile tip of the cusps after 3 days of culture. At day 7-8 the first dentin was observed. In contrast to the findings of Yamada et al. ( 1980) this dentin was van Kossa positive (calcium) Opposite to this dentin the first enamel matrix was found which showed a fine granular von Kossa staining. After 10 days of cultivation a thick layer of enamel
Fig. 1. Second day 2nd molar cultivated for 10 days. Toluidine blue, 1 pm epon section; a ameloblasts; em enamel matrix; p predentin: d dentin: o odontoblasts x 95 Received:
March
03OS-1651/8110807714J11$02.00/0
1981
771
1981
IN VITRO and J.H.M.WGltgens Vrije Universiteit ‘The Netherlands
matrix had been produced (Fig. 11, which was found to mineralize from the enamel dentin junction towards the ameloblasts (Fig. 2). In many of the cultured explants in the ameblasts in the bottom of the deep fissures an accumulation of secretory granules was found. Occasionally some local destruction was observed in these regions. apparently due to diffusion problems. All cultured explants (n = 85) were found to produce enamel matrix of the prismatic type in considerable amounts( 8-1099 of the total area). It can be concluded that for studying amelogenesis and mineralization, this culture system is a useful1 and reliable tool. References Slavkin, H C . ( 1979) Amelogenesis in vitro. Journal of Dental Research 58B, 735-741 Bronckers,A.L.J.J. and Wiiltgens,J.H.M(1980) Morphological and biochemical aspects of vit C deficiency during tooth germ development in vitro. Calcified Tissue International suppl 31, 13 Yamada,M., Bringas,P., Grodin,M .MacDougall, M and Slavkin, H . C . ( 1980) Developmental comparisons of murine secretory amelogenesis in viva, as xenografts on the chick chorio allantoic membrane and in vitro.Calcified Tissue International 31, 161-171
Fig 2. Detail left cusp. Von Kossa, lpm epon section: d dentin: em enamel matrix; a ameloblasts; si stratum intermedium; edj enamel dentin junction x 1025 Accepted:
June
0 1981 Academic
1981
Press Inc. (London)
Ltd.