Inhibition of tooth germ differentiation in vitro

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1 /. Embryo!, exp. Morph. Vol. 50, pp , 1979 Printed in Great Britain Company of Biologists Limited Inhibition of tooth germ differentiation in vitro By KIRSTI HURMERINTA, 1 IRMA THESLEFF 1 AND LAURI SAXEN 1 From the Department of Pathology, University of Helsinki, Finland SUMMARY Molar tooth germs from mouse embryos were studied in a Trowell-type organ culture. After 5 days of culture the odontoblasts had secreted predentine and the ameloblasts had differentiated. When cultured in the presence of /m. diazo-oxo-norleucine (DON), which is a glutamine analogue, the differentiation of odontoblasts was inhibited, but the teeth looked otherwise healthy. When DON was added after 2 days of culture in control medium (at this tims the odontoblasts in the cuspal area were already differentiated), it did not inhibit predentine secretion, ameloblast differentiation, nor enamel secretion. However, this was seen only in the cuspal area and the boundary to the undifferentiated, more cervical cells was distinct. The results support the concept that the mechanism of the differentiation of odontoblasts is different from that of the ameloblasts. We have shown earlier that a close association between the basement msmbrane and the mesenchymal cells is required for odontoblast differentiation. Because DON inteiferes with glycosaminoglycan and glycoprotein synthesis we suggest that DON inhibits odontoblast differentiation by affecting the mesenchymal cell surface and/or the basement membrane. INTRODUCTION The tooth-forming cells, the odontoblasts and the ameloblasts, differentiate in the bell-shaped tooth germ. The epithelial enamel organ surrounds the dental papilla, which is a condensation of mesenchymal cells derived from the neural crest. The epithelial and mesenchymal cells are separated from each other by a basal lamina, which is continuous during the early stages of differentiation (Reith, 1967; Silva & Kailis, 1972; Kallenbach, 1976; Meyer, Farbe, Staubli & Ruch, 1977). Differentiation of the cells occurs in an exact sequence. First the mesenchymal cells become polarized and start to secrete predentine. Thereafter the cells of the enamel epithelium differentiate into ameloblasts and secrete the organic matrix of enamel. Transfilter studies have indicated that the first step requires a close association between the interacting mesenchymal and epithelial cells (Thesleff, Lehtonen, Wartiovaara & Saxen, 1977). This association was localized to the interface 1 Authors' address: Department of Pathology, University of Helsinki, Haartmaninkatu 3, Helsinki 29, Finland.

2 100 K. HURMERINTA, I. THESLEFF AND L. SAXEN between the mesenchymal cells and the basement membrane under the enamel epithelium (Thesleff, Lehtonen & Saxen, 1978). As differentiation of the epithelial cells into the ameloblasts is preceded by predentine secretion by odontoblasts, this has been suggested as inducing ameloblasf differentiation (Ruch, Farbe, Karcher-Djuricic & Staubli, 1974). However, also at this stage the basal lamina is degraded and epithelial microvilli project through its discontinuities so making contact with the odontoblast cell processes (Kallenbach, 1971; Slavkin & Bringas, 1976; Meyer et al. 1977). These cell-to-cell contacts have been suggested as playing a role in the determination of ameloblasts (Slavkin & Bringas, 1976). The aim of this study was to examine the effects of the glutamine analogue diazo-oxo-norleucine (DON) on the differentiation of odontoblasts and ameloblasts. By blocking the utilization of glutamine in the transamidination reactions DON interferes with at least two important metabolic pathways: the synthesis of glycosaminoglycans (GAG) and glycoproteins (Ghosh, Blumenthai, Davidson & Roseman, 1960; Telser, Robinson & Dorfman, 1965) and the synthesis of purines (Buchanan, 1973). DON inhibits palatal epithelial cell adhesions in vitro apparently by blocking the synthesis and secretion of the surface-associated glycoproteins (Greene & Pratt, 1977). Furthermore, DON inhibits kidney tubule induction in the metanephric mesenchyme probably by blocking the synthesis of GAG and glycoproteins (Ekblom et al. 1979). Since these molecules are important constituents of the basement membrane and the cell surface, inhibition of their synthesis could also be expected to interfere with the differentiation of odontoblasts and ameloblasts. MATERIAL AND METHODS Tooth buds were dissected from 16-, 17- and 18-day-old hybrid mouse embryos, BALBc/CBA. The day of vaginal plug was designated as day 0. The lower jaw was removed and the first mandibular molars were dissected free from surrounding tissue in phosphate buffered saline. For recombination cultures the enamel organ was separated from the dental papilla by treatment with cold 2-25 % trypsin % pancreatin solution for 10 min and after 15min of incubation in culture medium at room temperature the two components were mechanically separated and recombined on Millipore filter. A Trowell-type organ culture was used, in which the explants were supported by a piece of TAWP-Millipore filter (thickness 25 /im and nominal pore size 0-8 jltm) on a metal grid. The medium consisted of BGJb medium (Difco) supplemented with 20 % horse serum (Flow laboratories), 10 % chick embryo extract and 0-9 mm ascorbic acid (Thesleff, 1976). According to the Difco formula BGJb contains 1-37 mm glutamine but because our medium had been stored in a liquid form for approximately 1 year, most of the glutamine should have decomposed. According to Tritsch & Moore (1962) a 30 % reduction takes place in 30 days at 4 C.

3 Inhibition of tooth differentiation by DON 101 The culture dishes were kept in a humidified incubator at 37 C in an atmosphere of 5 % CO 2 in air. The explants were cultured for 5-14 days with daily changes of the medium. The explants were fixed in Zenker's solution, embedded in Tissue prep (Fisher Scientific Company, Fair Lawn, New Jersey) and serially sectioned at 7 /tm. They were stained with Mallory's phosphotungstic acid-haematoxylin, which stains the predentine red and the enamel matrix dark bluish-black. 6-Diazo-5-oxo-L-norleucine (DON) and amino imidazole carboxamide (A1C) were gifts from Dr Robert M. Pratt, National Institute of Dental Research, N1H, Bethesda, Maryland. DON was used in concentrations of JLLM and AIC at a concentration of 10 mm. Glutamine and glucosamine were obtained from Sigma Chemical Company, St Louis, Missouri, and they were used at a concentration of 10 mm. RESULTS (a) Differentiation of odontoblasts The mandibular first molar of a 16-day-old mouse embryo is in the early bell stage of development. The epithelial and mesenchymal cells are histologically undifferentiated. After 6 days of culture the mesenchymal cells had differentiated into odontoblasts, which secreted predentine and the onset of ameloblast differentiation was also seen frequently (Fig. 1A, Table 1). DON (10-50 jm) inhibited odontoblast differentiation. Predentine secretion was not seen in any of the 20 tooth germs grown in the presence of DON (Fig. IB). Even the explants grown in 50 JLIM DON appeared viable by light microscope examination (see Fig. 2D). Even in 17-day-old tooth germs no histological sign of differentiation is seen. After 5 days of culture in control medium predentine had regularly been secreted in a continuous layer extending from one cusp to another. At this time polarization of ameloblasts was frequently seen and sometimes enamel matrix had been secreted by the ameloblasts (Fig. 1C). In 27 of the 46 tooth germs grown in the presence of DON (10-50 /LM) no predentine secretion was seen (Fig. ID). In 19 tooth germs a small amount of predentine had been secreted at the cuspal tips. In these explants a distinct boundary was noted between the predentine secreting odontoblasts and the undifferentiated more cervical mesenchymal cells. In 18-day-old tooth germs the mesenchymal cells in the cuspal area are aligned and this we consider the first sign of differentiation. DON (10-50 JLIM) did not inhibit predentine secretion by the odontoblasts in the cuspal area. However, the differentiated area was restricted to the cuspal tips so that there was a distinct boundary between the tips and the undifferentiated more cervical area (Table 1). In the control explants predentine had always been secreted as a continuous layer extending from one cusp to another.

4 102 K. HURMERINTA, I. THESLEFF AND L. SAXEN Fig. 1. Photomicrographs illustrating the effect of DON on mouse molar tooth germs in vitro. Mallory's phosphotungstic acid-haematoxylin stain. (A) A 16-dayold tooth germ cultured for 6 days in control medium. Predentine (PD) has been secreted by differentiated odontoblasts. Ameloblasts have started to polarize. (B) A 16-day-old tooth germ cultured for 6 days in the presence of 10 /m DON. Mesenchymal cells are undifferentiated. (C) A 17-day-old tooth germ cultured for 5 days in control medium. The differentiated odontoblasts have secreted predentine and ameloblasts have polarized and started the secretion of enamel matrix. (D) A 17-day-old tooth germ cultured for 5 days in the presence of 30 /.CM DON. No sign of differentiation of mesenchymal cells (M) into odontoblasts is seen. M, mesenchymal cells; O, odontoblasts; PD, predentine; A, ameloblasts; EM, enamel matrix.

5 Inhibition of tooth differentiation by DON 103 Table 1. Effect of 30 JM DON on odontoblast differentiation in 16-, 17- and 18-day-old tooth germs after 5 or 6 days of culture Number of explants showing predentine (PD) secretion, used as a criterion for differentiation Age 16-day 17-day 18-day PD NoPD PD NoPD PD No PD Control DON30/tM 0 7 7* 13 4* 0 * The borderline between the differentiated cuspal area and the undifferentiated more cervical area was distinct. (b) Differentiation of ameloblasts The effect of DON on ameloblast differentiation and the secretion of enamel matrix was studied in experiments in which 17-day-old tooth germs were precultivated for 2 days in control medium and then for 7 days in the presence of DON (10-30 fim). After the first 2 days the mesenchymal cells in the cuspal area had already aligned (Fig. 2A) and occasionally secreted predentine. In the control teeth, which were cultured for the whole 9-day period without DON, a thick layer of predentine was formed and the epithelial cells had differentiated into ameloblasts and frequently secreted enamel matrix (Fig. 2B). In the cuspal area of DON-treated tooth germs an equal amount of predentine had been secreted. Ameloblasts had differentiated in all 16 explants and enamel matrix had been secreted in 13 of them (Fig. 2C). However, a distinct demarcation line was observed between the odontoblasts secreting predentine and the undifferentiated mesenchymal cells (Fig. 2D). (c) Culture of recombined epithelium and mesenchyme In explants in which the epithelium and mesenchyme of 17-day-old tooth germs were separated and subsequently recombined, predentine had always been formed after 5 days of culture (Fig. 3 A). DON (10-30 /m) inhibited the secretion of predentine, used as a criterion for odontoblast differentiation, in all 20 explants (Fig. 3B). (d) Reversibility and prevention of the DON effect When 16-day-old tooth germs were transferred to control medium after 7 days of culture in the presence of DON (10 JUM), recovery was regular. After 5 days of subculture, predentine and enamel matrix had been secreted in all three explants (Fig. 4 A). The inhibitory effect of DON on odontoblast differentiation was fully over-

6 104 K. HURMERINTA, I. THESLEFF AND L. SAXEN c Fig. 2. Photomicrographs illustrating the effect of DON on the 17-day-old tooth germs precultured in control medium for 2 days. Mallory's phosphotungstic acidhaematoxylin stain. (A) The tooth germ after 2 days of preculture in control medium. Mesenchymal cells have aligned under the epithelium at the cuspal tips. (B) A tooth germ cultured for 9 days in control medium. Odontoblasts have secreted predentine and ameloblasts have formed enamel matrix. (C) A tooth germ precultured for 2 days in control medium and 7 days in the presence of 30 /xu DON. Predentine has been secreted by the odontoblasts and enamel matrix by the ameloblasts. (D) The epithelial-mesenchymal interface in the intercuspal area of a similar explant as in C. DON concentration is 50 /*M. A distinct boundary (arrow) is seen between the differentiated and undifferentiated area. M, mesenchymal cells; O, odontoblasts; PD, predentine; A, ameloblasts; EM, enamel matrix.

7 Inhibition of tooth differentiation by DON 105 Fig. 3. Recombined explants of the epithelial and mesenchymal components of 17-day-old tooth germs cultured for 4 days. Mallory's phosphotungstic acidhaematoxylin stain. (A) In control medium the mesenchymal cells have differentiated into odontoblasts and secreted predentine. (B) In the presence of 10 /*M DON the mesenchymal cells have not differentiated. M, mesenchymal cells; O, odontoblasts; PD, predentine.

8 106 K. HURMERINTA, I. THESLEFF AND L. SAXEN * Fig. 4. Photomicrographs illustrating the reversibility and prevention of the DON effect on the 16-day-old molar tooth germs in vitro. Mallory's phosphotungstic acid-haematoxylin stain. (A) A tooth germ precultured in the presence of 10 fiu DON for 7 days and subsequently in control medium for 5 days. Predentine has been secreted by the odontoblasts and enamel matrix by the ameloblasts. (B) A tooth germ cultured for 7 days in the presence of 10 [M. DON and 10 mm glutamine. The odontoblasts have secreted predentine and the ameloblasts are polarized. O, odontoblasts; PD, predentine; A, ameloblasts; EM, enamel matrix. ridden by the excess of glutamine. In all four 16-day-old tooth germs cultured in the presence of DON (10/AM) and glutamine (10 mm) for 7 days predentine had been secreted and ameloblasts were polarized (Fig. 4B). Neither glucosamine nor the purine analogue, amino imidazol carboxamidine (AIC) overrode the effect of DON on odontoblast differentiation. Glucosamine (10 mm) alone did not inhibit normal development in the four 16-day-old tooth germs examined, but when both glucosamine (10/AM) and DON (10/*M) were present the survival of most of the eight explants was poor. When the 16-day-old tooth germs were cultured in the presence of AIC (10 mm) normal differentiation was prevented in all the four explants. When both AIC (10 mm) and DON (10 /.IM) were present odontoblast differentiation was not observed in the three explants examined. DISCUSSION Our results show that in the developing tooth diazo-oxo-norleucine (DON) inhibits the differentiation of odontoblasts but not that of the ameloblasts. Furthermore, no effect on the secretion of predentine and enamel matrix could be observed in light microscopy. In tooth germs of 16-day-old mouse embryos

9 Inhibition of tooth differentiation by DON 107 DON completely inhibited odontoblast differentiation and subsequent secretion of predentine. In 17-day-old tooth germs odontoblast differentiation was inhibited in half of the explants whereas in the rest predentine had been secreted in the cuspal area. We suggest that the cuspal cells had already been determined in vivo prior to the explantation although histological differentiation could not be observed. This suggestion is supported by the results of culture of more advanced tooth germs. Predentine was seen in the cuspal area of all 10 DON treated 18-day-old tooth germs. Furthermore, in explants in which the cuspal odontoblasts had been allowed to differentiate over 2 days of culture prior to DON addition, differentiation proceeded only in the cuspal area, and the boundary with the undifferentiated more cervical area was clear-cut. In recombination cultures of 17-day-old tooth germs DON inhibited odontoblast differentiation in all explants. This was probable due to the loss of basement membrane during enzymic separation of the epithelium and the mesenchyme (Thesleff et ah 1978), resulting in loss of the possible existing alignment of mesenchymal cells at the cuspal tips. Thus it seems that DON had an effect only on undifferentiated mesenchymal cells and that it did not affect predentine secretion by already determined odontoblasts. That DON did not affect secretion of predentine could have been expected since 99 % of predentine is collagen (Eastoe, 1967), and it has been reported that DON does not inhibit collagen synthesis (Bhatnager & Rapaka, 1971). Agents interfering with collagen synthesis have been shown to inhibit tooth morphogenesis (Galbraith & Kollar, 1974; Ruch et ah 1974). The actual effect of these substances on odontoblast differentiation is, however, difficult to evaluate since they have obviously prevented the secretion of predentine. Thus it has not been possible to distinguish the effect of these agents on the epitheliomesenchymal interaction leading to odontoblast differentiation from their obvious effect on the expression of the differentiated state of these cells, i.e. on secretion of predentine. Collagen may well play an important role in these interactive events (Hetem, Kollar, Cutler & Yaeger, 1975) but it is improbable that the collagen molecule itself would induce differentiation. Attempts to induce odontoblast differentiation by various collagen substrates have failed (Koch, 1975; Thesleff, 1978). DON interferes with the synthesis of GAG and glycoproteins by inhibiting the glutamine dependent conversion of fructose-6-phosphate to glucosaminophosphate (Ghosh et ah 1960; Telser et ah 1965). DON also combines irreversibly with the enzyme necessary for purine nucleoside synthesis (Buchanan, 1973). An excess of glutamine overrode the effect of DON on odontoblast differentiation. The purine analogue AIC did not override the effect of DON. This result is in accordance with the reports of Greene & Pratt (1977) and Ekblom et a]. (1979). Glucosamine, which would be expected to overcome the block on GAG and glucoproteins synthesis (Telser et ah, 1965) did not normalize development. This may depend on a toxic effect of glucosamine on embryonic

10 108 K. HURMERINTA, I. THESLEFF AND L. SAXEN tissue (Kim & Conrad, 1974). Similar results have been reported in other studies as well (Ekblom et al. 1979). Therefore, these experiments are not contradictory to the conclusion that odontoblast differentiation may depend on GAG and glycoprotein synthesis. The mechanism of the DON-resistant induction of ameloblasts seems to be different from that of the odontoblasts, because the same DON concentrations which inhibited the differentiation of odontoblast had no effect on that of ameloblasts. For ameloblast determination actual contacts between, the epithelial and the mesenchymal cells have been considered necessary (Kallenbach, 1971; Slavkin & Bringas, 1976) as in the induction of metanephric kidney tubules (Saxen et al. 1976). The latter has, however, been shown to be DONsusceptible (Ekblom et al. 1979). This suggests that either the two contactmediated interactions operate via different mechanisms, or, that ameloblast differentiation is determined by compounds of the extracellular matrix. Since our results seem to exclude the significance of extracellular GAGs and glycoproteins, collagen might still be considered as a possible mediator of the inductive signal for ameloblast differentiation (Ruch et al. 1974). Results of our transfilter studies have suggested that odontoblast differentiation is preceded by a close contact between mesenchymal cell processes and the basement membrane on the epithelial side (Thesleff et al. 1978). We suggest that the inhibitory effect of DON on odontoblast differentiation was due to its effect on the structure and constitution of the basement membrane and/or of the mesenchymal cell periphery. The changed structure may not allow the transfer of inductive signals which are normally mediated via contacts between cells and extracellular matrix. We wish to thank Dr Robert M. Pratt, National Institute of Dental Research, NIH Bethesda, Maryland, for providing DON and AIC. This investigation was supported by the Emil Aaltonen Foundation and by the Finnish Dental Society. REFERENCES BHATNAGAR, R. S. & RAPAKA, S. S. R. (1971). Cellular regulation of collagen biosynthesis. Nature, New Biol. 234, BUCHANAN, J. M. (1973). The amidotransfereases. In Advances in Enzymology (ed. A. Meister), 39, New York: John Wiley & Sons. EASTOE, J. E. (1967). Chemical organization of the organic matrix of dentine. In Structural and Chemical Organization of Teeth (ed. A. E. W. Miles), 2, New York: Academic Press. EKBLOM, P., LASH, J. W., LEHTONEN, E., NORDLING, S. & SAXEN, L. (1979). Inhibition of morphqgenetic cell interactions by 6-diazo-5-oxo-norleucine (DON). Expl Cell Res. (In the Press.)" GALBRAITH, D. B. & KOLLAR, E. J. (1974). Effects of L-azatidine-2-carboxylic acid, a proline analogue, on the in vitro development of mouse tooth germs. Archs oral Biol. 19, 1 Hill 76. GHOSH, S., BLUMENTHAL, H. J., DAVIDSON, E. & ROSEMAN, S. (1960). Glucosamine metabolism. V. Enzymatic synthesis of glucosamine 6-phosphate. /. biol. Chem. 235,

11 Inhibition of tooth differentiation by DON 109 GREENE, R. M. & PRATT, R. M. (1977). Inhibition by diazo-oxo-norleucine (DON) of rat palatal glycoprotein synthesis and epithelial cell adhesion in vitro. Expl Cell Res. 105, HETEM, S., KOLLAR, E. J., CUTLER, L. S. & YAEGER, J. A. (1975). Effect of a-a-dipyridyl on the basement membrane of tooth germs in vitro. J. dent. Res. 54, KALLENBACH, E. (1971). Electron microscopy of the differentiating rat incisor ameloblast. /. Ultrastruct. Res. 35, KALLENBACH, E. (1976). Fine structure of differentiating ameloblasts in the kitten. Amer. J. Anat. 145, KIM, J. J. & CONRAD, H. E. (1974). Effect of D-glucosamine concentration on the kinetics of mucopolysaccharide biosynthesis in cultured chick embryo vertebral cartilage. /. biol. Chem. 249, KOCH, W. E. (1975). In vitro development of isolated tooth tissues on collagenous substrates. /. dent. Res. 54, 137. MEYER, J. M., FARBE, M., STAUBLI, A. & RUCH, J. V. (1977). Relations cellulaires au cours de l'odontogenese. /. biol. Buccale 5, REITH, E. J. (1967). The early stage of amelogenesis as observed in molar teeth of young rats. /. Ultrastruct. Res. 17, RUCH, J. V., FARBE, M., KARCHER-DJURICIC, V. & STAUBLI, A. (1974). The effects of L- azetidine-2-carboxylic acid (analogue of proline) on dental cytodifferentiations in vitro. Differentiation 2, SAXEN, L., LEHTONEN, E., KARKINEN-JAASKELAINEN, M., NORDLING, S. & WARTJOVAARA, J. (1976). Are morphogenetic tissue interactions mediated by transmissible signal substances or through cell contacts? Nature, Lond. 259, SILVA, D. G. & KAILIS, D. G. (1972). Ultrastructural studies on the cervical loop and the development of the amelo-dentinal junction in the cat. Archs oral Biol. 17, SLAVKIN, H. C. & BRINGAS, P. (1976). Epithelial-mesenchyme interactions during odontogenesis. IV. Morphological evidence for direct heterotypic cell-cell contacts. Devi. Biol. 50, TELSER, A., ROBINSON, H. C. & DORFMAN, A. (1965). The biosynthesis of chondroitin-sulfate protein complex. Proc. natn Acad. Sci., U.S.A. {Wash.). 54, THESLEFF, 1. (1976). Differentiation of odontogenic tissues in organ culture. Scand. J. dent. Res. 84, THESLEFF, ]., LEHTONEN, E., WARTIOVAARA, J. & SAXEN, L. (1977). Interference of tooth differentiation with interposed niters. Devi Biol. 58, THESLEFF, I., LEHTONEN, E. & SAX N, L. (1978). Basement membrane formation in transfilter tooth culture and its relation to odontoblast differentiation. Differentiation 10, THESLEFF, I. (1978). The role of the basement membrane in odontoblast differentiation. /. biol. Buccale 6, TRITSCH, G. L. & MOORE, G. E. (1962). Spontaneous decomposition of glutamine in cell culture media. Expl Cell Res. 28, {Received 10 July 1978, revised 30 October 1978) EHB 50

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