Scand. J. Dent. Res. 1976: 84: 353-356 (Key words: amelogenesis; dentinogenesis; odontogenesis; organ culture)

Differentiation of odontogenic tissues in organ culture IRMA THESLEFF / / / Department of Pathology, University of Helsinki, Helsinki, Finland ABSTRACT - Molar tooth germs from 17-d-old mouse embryos were cultivated in a Trowelltype culture, and different culture media were tested for their ability to support enamel formation. The medium which allowed secretion of considerable amounts of enamel matrix by ameloblasts consisted of BGJb medium supplemented with 20 % horse serum, 10 % chick embryo extract and 0.9 mM ascorbic acid. At the onset of culture the teeth were in the early bell stage. After 2 weeks of cultivation both odontoblasts and ameloblasts had differentiated, and considerable amounts of predentin and enamel matrix had been secreted. Similar development was also seen in teeth which had been enzymatically separated into the mesenchymal dental papilla and epithelial enamel organ and subsequently recombined in vitro. This method allows good differentiation of odontogenic tissues, and is considered suitable for further studies of tissue interactions in the tooth rudiment.

Tooth germs were cultivated in vitro already in the 193O's (GLASSTONE 1936), and numerous organ culture techniques have since been applied to studies on tooth development of various species (SzABO 1954, HAY 1961, POURTOIS 1964, KOCH 1965, WIGGLES WORTH 1968,

RUCH,

& MOULLEC 1970). However, most of the methods described have either not allowed enamel matrix secretion by the ameloblasts or this secretion has occurred only occasionally (GLASSTONE 1965). Constant enamel formation in organ culture has so far been reported only by KOCH (1965) and WiGGLESWORTH (1968). The aim of this study was to find suitable culture conditions for future studies on tissue interactions and differentiation of odontogenic mesenchyme and epithelium. Because secretion of enamel matrix KARCHER-DJURICIC

can be regarded as a definite criterion for ameloblast differentiation, the culture conditions should allow enamel formation. The method reported here is largely based on the two previous studies with successful enamel formation (KOCH 1965, WIGGLESWORTH 1968), and it supports regularly the formation of considerable amounts of enamel matrix.

Material and methods Tooth buds were taken from 17-d-old hybrid mouse embryos, BALBc/CBA. The day of vaginal plug was taken as day 0. The mandible was removed and the mandibuiar first molars were dissected free from surrounding tissues in PBS. For separation of the enamel organ from the dental papilla the teeth were placed in cold 2.75 % pancreatin-trypsin solution for 10 min and the two components were then separated mechanically at room temperature. A Trowell-type organ culture was used, in

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Fig. 1. Mandibuiar first molar tooth rudiment of 17-d-old mouse embryo at start of cultivation. At higher magnification of epithelio-mesenchymal interface no differentiation in cells of the dental papilla or of the inner enamel epithelium can be observed. which the explants were grown at the mediumgas interface and supported by a piece of Millipore® filter on a metal grid. The medium allowing enamel formation consisted of BGJb (BiGGERS, GwATKiN & HEYNER 1961) Supplemented with 20 % horse serum (Flow Laboratories), 10 % chick embryo extract, and 0.9 mM ascorbic acid. Other media for which results are reported here were BGJb without protein supplement and with or without 0.9 mM ascorbic acid, and MEM (Eagle's minimum essential medium in Earle's balanced salt solution) -I- 10 % fetal calf serum (Flow Laboratories), and 0.9 mM ascorbic acid. In all cultures the medium was changed every 2 d, and the culture dishes were kept in a humidified incubator at 37 °C in an atmosphere of 5 % CO2 in air. After 3—14 d of cultivation the explants were fixed in Zenker's fluid, embedded in paraffin and serially sectioned at 6 jxm. They were stained with Mallory's phosphotungstic acidhematoxylin, which stains predentin red and enamel matrix bluish black, thus allowing easy detection of enamel secretion. I Results

The mandibuiar first molar of the 17-dold mouse embryo is at its early bell stage of development and the mesenchymal cells have not yet differentiated to odontoblasts (Fig. 1). When these tooth germs were cultivated for 2 weeks in the chemically

defined BGJb medium, odontoblasts regularly differentiated and secreted predentin. Ascorbic acid stimulated the secretion of this collagenous matrix. Polarization of ameloblasts, however, occurred only infrequently, and enamel matrix secretion was not seen. When cultivated in the presence of serum (MEM -|- 10 % fetal calf serum) differentiation occurred more rapidly and proceeded to a more advanced stage. Ameloblast polarization was always observed after 1 week of culture, but enamel matrix was not formed even after 2 weeks of culture. The medium which allowed constant secretion of enamel matrix consisted of BGJb medium, 20 % horse serum, 10 % chick embryo extract, and 0.9 mM ascorbic acid. Lower concentrations of serum or embryO' extract also allowed enamel formation occasionally, but only to a lesser extent. Differentiation of tooth rudiments in this protein-rich medium occurred about two times slower than in vivo. Secretion of predentin, which in vivo starts on the 19th day of development, was seen after 4 d in culture, and enamel formation, starting on the 20th day of in vivo development, was detected after 1 week

TOOTH DIFFERENTIATION IN VITRO

355

Fig. 2. A rudiment like the one in Fig. 1 after 2 weeks of cultivation in BGJb medium supplemented with 20 % horse serum, 10 % chick embryo extract, and 0.9 mM ascorbic acid. Considerable amounts of enamel matrix have been secreted by the ameloblasts. Higher magnification shows the appearance of tall polarized ameloblasts (A) and odontoblasts (O), and their respective secretory products, enamel matrix (EM) and predentin (PD). Mallory's phosphotungstic acid-hematoxylin stain.

of cultivation. After 2 weeks in organ culture considerable amounts of enamel matrix had been formed, and the explant appeared healthy (Fig. 2). In explants in which the epithelium and mesenchyme of the 17-d-old tooth rudiment had been enzymatically separated prior to cultivation, and subsequently recombined in vitro, differentiation occurred as in whole rudiments. The exact sequentiality of differentiation, starting

from odontoblast differentiation and ending with enamel matrix secretion was seen here too, and after 2 weeks of cultivation considerable amounts of enamel matrix had been formed (Fig. 3). Discussion

The results of this study show that the degree of differentiation of tooth rudiments achieved in organ culture depends

Fig. 3. A recombinant of enzymatically dissociated molar mesenchyme and epithelium of a 17d-old embryo after 2 weeks of cultivation in the same medium as the whole rudiment in Fig. 2.

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on culture conditions. Odontoblasts differentiated and secreted predentin even in chemically defined medium, whereas differentiation of ameloblasts occurred more readily when serum was added to the medium. For the secretion of enamel matrix by the anaeloblasts embryo extract in addition to high serum concentration ( 2 0 % ) proved essential. The requirements for full functional maturation of ameloblasts are not known. Secretion of enamel matrix by ameloblasts occurs readily when tooth germs are grown as transplants, e.g. in the abdominal wall (HuGGiNS, MCGARROLL & DAHLBERG 1934), in the anterior chamber of

the

eye

(FLEMING

1952,

KOLLAR

&

1970) or in the testis (HERITIER 1970). Also, when cultured as grafts on the chick chorioallantoic membrane, teeth develop into the stage of enamel matrix secretion (SLAVKIN & BAVETTA 1968). As stressed above, in most previous studies in vitro this degree of dental development has only occasionally been achieved, and it has often been concluded that a high protein content is necessary for the secretoi-y function of ameloblasts (GLASSTONE 1965, KOCH 1972). In the culture conditions used in the present study, the enzymatically dissociated dental papilla mesenchyme and enamel organ epithelium retained their ability to differentiate and form enamel matrix. Thus this method allows experimental manipulation of the tooth rudiment and can be used for studies on the epithelio-mesenchymal interaction in tooth development. BAIRD

References BiGGERs, J. D., GwATKiN, R. B. L. & HEYNER, S.: The growth of avian and mammalian tibiae on a relatively simple chemically defined medium. Exp. Cell Res. 1961: 25: 41-58. FLEMING, H . S.: Homologous and heterologous intraocular growth of transplanted tooth germs. / . Dent. Res. 1952: 31: 166-188.

S.: The development of tooth germs in vitro. J. Anat. 1936: 70: 260-266. GLASSTONE, S.: The development of tooth germs in tissue culture. In: WILLMEN, E . N . (ed.): Cell and tissues in culture. Vol. 2. Academic Press, New York 1965, p. 273. HAY, M . F . : The development in vivo and in vitro of the lower incisor and molars of the mouse. Arch. Oral Biol. 1961: 3: 86-109. HERITIER, M . : Interet de la transplantation intra-testiculaire pour l'etude de la differenciation des ebauches dentaires chez la souris. Rec. Stomatol. Odontol. NF. 1970: 98: 7990. HuGGiNs, C. B., McC.\RROLL, H. R. & DAHLBERG, A. A.: Transplantation of tooth germ elements and the experimental heterotopic formation of dentine and enamel. / . Exp. Med. 1934: 60: 199-210. KOCH, W . E . : In vitro development of tooth rudiments of embryonic mice. Anat. Rec. 1965: 152: 513-524. KOCH, W . E . : Tissue interaction during in vitro odontogenesis. In: SLAVKIN, H . C. & GLASSTONE,

BAVETTA, L . A. ( e d . ) : Developmental

aspects

of oral biology. Academic Press, New York 1972, p. 151. KOLLAR, E . J. & BAIRD, G.: Tissue interactions in embryonic mouse tooth germs. II. The inductive role of the dental papilla. / . Embryol. Exp. Morphol. 1970: 24: 173-186. POURTOIS, M . : Comportement en culture in vitro des ebauches dentaires de rongeurs prelevees aux stades de predifferenciation. / . Embryol. Exp. Morphol. 1964: 12: 391-406. RUCH, J. V., KARGHER-DJURICIC, V. & MOUL-

LEc, N.: Etude de la differenciation in vitro d'ebauches de molaires inferieures d'embryons de souris. C. R. Soc. Biol. 1970: 164: 419-422. SLAVKIN, H . C. & BAVETTA, L . A.: Organogenesis: Prolonged differentiation and growth of tooth primordia on the chick chorio-allantois. Experientia 1968: 24: 192-194. SzABO, G.: Studies on the cultivation of teeth in vitro. J. Anat. 1954: 88: 31-45. WiGGLESWORTH, D. J.: Formation and mineralisation of enamel and dentine by rat tooth germs in vitro. Exp. Cell Res. 1968: 49: 211-215. Address: III. Department of Pathology University of Helsinki SF-00290 Helsinki 29 Finland

Differentiation of odontogenic tissues in organ culture.

Molar tooth germs from 17-d-old mouse embryos were cultivated in a Trowell-type culture, and different culture media were tested for their ability to ...
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