Tissue Interactions in Tooth Development

Differentiation

Differentiation (1981) 18: 75-88

0 Springer-Verlag 1981

Review Articles

Tissue Interactions in Tooth Development IRMA THESLEFF* and KIRSTI HURMERINTA Department of Pathology and Department of Pedodontics and Orthodontics, University of Helsinki, Haartmaninkatu 3, 00290 Helsinki 29, Finland

Introduction Tooth development involves complex morphogenetic movements of mesenchymal and epithelial cells, followed by differentiation of tooth-specificsecretory cells. These cells, the odontoblasts and ameloblasts, form the organic matrices of the mineralizing dentin and enamel, respectively. The early stages of tooth morphogenesis are comparable to those of other integumental derivatives like feathers, hairs, and glands. Later differentiative events, involving not only enamel organ morphogenesis but also mesenchymal and epithelial cell differentiation and matrix secretion, are characteristic of tooth development. Morphogenesis and cell differentiation are controlled by a chain of interactive events between the epithelial and mesenchymal tissues. This has rendered the developing tooth an appropriate model system for studies on different aspects of epitheliomesenchymal interactions during embryonic development. The advancing development of a given organ can generally be viewed as a multistep process in which a chain of tissue interactions controls differentiation [70,72,76]. These sequential interactions are usually also reciprocal, i.e., the target tissue affects in turn the differentiation of the inductor tissue. As the cells gradually assume a higher level of differentiation,the number of options for their further differentiation pathways decreases. It has been suggested that the early interactions guiding the differentiation of uncommitted cells are basically directive in nature, whereas the later interactions are merely permissive. Thus, the function of the ‘inductor’ tissue during the permissive interactions is to create suitable conditions To whom correspondence should be addressed

in which the target tissue can express its differentiated state. The tissue interactions during tooth development seem to comply well with these general principles. The schematic presentation in Fig. 1 shows that the ectomesenchymal neural crest cells and the stomatodeal epithelial cells gradually acquire higher levels of differentiation and end up as secretory odontoblasts and ameloblasts, respectively. The tissue interactions are definitely sequential and reciprocal in nature, and as recently pointed out by Kollar [43], the critical directive events that permit the expression of highly differentiated activity in the cells may take place already during the early phases of development. The scheme in Fig. 1, in which the suggested consecutive interactive steps are represented by numbered arrows, serves merely to simplify the following discussion on the complex tissue interactions that control tooth development.

The Chain of Interactions Tooth development has been divided into three partially overlappingphases, the periods of initiation, morphogenesis, and cell differentiation [43]. In Fig. 1 arrows 1-3 indicate interactions controlling initiation, arrows 4 and 5 those controlling morphogenesis, and arrows 6-8 the interactions involved in cell differentiation. Initiation of tooth formation implies determination of the dental mesenchyme and formation of the epithelial dental lamina. The results of the earliest experimental studies on tissue interactions in tooth development indicated that the mesenchymal component of the developing tooth is of neural crest origin, and that these ectomesenchymal cells are the

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I. Thesleff and K.Hurmerinta: Tissue Interactions in Tooth Development

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Fig.1. Simplified schema of suggested tissue interactions (arrows 1-8) guiding tooth development. As a result of the sequential and reciprocal interactions, the mesenchymal cells (presumably of neural crest origin) and the oral epithelial cells acquire higher levels of differentiation, which is terminally expressed as predentin and enamel secretion by the differentiated odontoblasts and ameloblasts (modified from [a])

primary factor in initiating tooth development and in controlling epithelial morphogenesis [181. These studies were done on amphibian and fish embryos, and involved transplantation of various tissue components or combinations. In mammalian species it has still not been definitely shown that tooth mesenchyme originates from the neural crest. The dental mesenchymal cells which differentiate into odontoblasts represent one type of skeletogenic cells, as their differentiation is also expressed by secretion of a collagenous, mineralizing extracellular matrix (ECM). Thus, their determination may have characteristics similar to those of the chondrogenic and osteogenic neural crest cells. The earliest cell interactions controlling determination of dental mesenchyme have probably already taken place during the migration of neural crest cells (Fig. 1, arrow 1). Studies on avian embryos have indicated that the pathway along which neural crest cells migrate determines the nature of their subsequent differentiation [48]. There is evidence that differentiation of the neural crest cells giving rise to various skeletal tissues in the craniofacial region is controlled by interactions of the migrating cells with

other cells and ECM on their migratory route (551. The final site where these neural crest cells localize also affects their differentiation. This has been established for maxillary and mandibular mesenchyme cells whose osteogenic potential and subsequent membrane bone formation depend on interactions with maxillary and mandibular epithelium [102, 1031. Also chondrogenic differentiation of ectomesenchymal cells forming scleral cartilages and secondary cartilages in the maxilla depends on local epigenetic factors at their final position [57, 991. It has not been established whether the oral epithelium influences the determination of dental mesenchymal cells (Fig. 1. arrow 2). The epithelium obviously controls mesenchymal cell differentiation at a later stage of tooth development (see below), but due to the sequential tissue interactions, it has been difficult to study the actual role of oral ectoderm during the initiative phase. The factors controllingthe definitive sites of tooth buds have been frequently discussed [5,18,59]. It has not, however, been established whether the information on the sites of the dental laminae resides in the epithelial tissue itself, or whether they are determined by the underlying stroma. It has been proposed that the developing capillaries or nerves determine the position of the tooth buds [17,43,63]. In a recent study the nerves could be traced to the last mesenchymal cell under the epithelial cells of the future tooth bud [43]. The capillaries and nerves have been suggested either to function as tracts guiding the migration of neural crest cells, or to trigger directly the formation of the dental laminae. During the rnorphogenetic phase of tooth development, the form of the tooth is established by morphogenetic movements of the tissues. The epitheliomesenchymal interface undergoes folding whereby the cuspal pattern of the tooth and the future dentinoenamel junction is established (Fig. 2A and B). The epithelial component develops into an enamel organ composed of characteristic cell types. Early transplantation studies already demonstrated that the morphogenesis of the mammalian tooth depends on epitheliomesenchymal interactions [181

Fig. 2. Light micrographs of mouse molar tooth germs at different developmental stages. A Bud stage. The epithelial tooth bud has invaginated the underlying mesenchyme. An area of condensed mesenchymal cells can be distinguished under the bud. B Early bell stage. The epithelial component, now called the enamel organ, has grown around the condensation of the mesenchymal cells, the dental papilla. The mesenchymal cells and the cells of the enamel epithelium appear undifferentiated. C-F Higher magnifications of the epithelio-mesenchymalinterface, illustrating cell differentiation in more advanced bell stage tooth germs. C The mesenchymalcells directly underlining the enamel epithelium have aligned under the epithelium. D The polarized odontoblasts have initiated predentin secretion (arrow). E More predentin has been secreted and the epithelial cells have polarized into ameloblasts. F The ameloblasts have secreted enamel matrix. Abbreviations used are: E epithelium, M mesenchyme, EE enamel epithelium, 0 odontoblasts, PD predentin, A ameloblasts, EM enamel matrix

I. Thesleff and K. Hurmerinta: Tissue Interactions in Tooth Development

Fig. 2. (Legend see p. 76)

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Fw. 3. Results of reciprocal combinations of gingival epithelium and dental mesenchyme (A), and dental epithelium and gingival mesenchyme (B). A The gingival epithelial cells have differentiatedinto ameloblasts and secreted enamel matrix (arrow). B The dental epithelium shows squamous metaplasia with keratinization, but no differentiation into ameloblasts. Abbreviations: E epithelial cells, M mesenchymal cells, 0 odontoblasts, PD predentin, A ameloblasts

(Fig. 1, arrows 4 and 5). From the studies by Kollar’s group, it became evident that the mesenchymal tissue instructs epithelial morphogenesis [42]. It was demonstrated that transplants of reciprocal combinations of the mesenchymal and epithelial components of incisor and molar tooth germs acquired the typical incisive or molar shape according to the origin of the mesenchymal tissue. It was also shown that the dental mesenchyme was able to induce the formation of typical enamel organs in nondental epithelia, e.g., foot pad epithelium, and that these epithelial cells differentiated into ameloblasts which secreted enamel matrix [42]. These observations have since been confirmed in other studies [66,91], and the results of one such experiment are presented in Fig. 3. Here reciprocal combinations of dental and gingival epithelium and mesenchyme from mouse embryos were cultured in vitro, and it was shown that gingival epithelium differentiated into ameloblastswhen combined with dental mesenchyme, whereas dental epithelium keratinized in combination with gingival mesenchyme. Recent experiments by Kollar’s group

have indicated that the dental mesenchyme from mouse embryos is able to induce enamel organ formation and ameloblast differentiation even in chick epithelium. The authors suggest that the absence of enamel synthesis in Aves is due to alterations in the tissue interactions required for odontogenesis, and not to a loss of genetic information [44]. Tooth morphogenesis can be inhibited by agents that disrupt the ECM between the epithelial and mesenchymal tissues. It has been shown that unaffected collagen deposition and glycoconjugate synthesis are prerequisites for normal tooth morphogenesis [14,26, 29, 30, 67, 97, 981 (see also p. 83). The branching morphogenesis of the developing salivary gland has been shown to depend on the ECM at the epitheliomesenchymal interface [4]. Presumably the ECM serves to stabilize the pattern of epithelial morphogenesis in a similar way during tooth development. The final cell &fleerentiation takes place during the bell stage of tooth development (Fig. 2C-F), and at

I. Thesleff and K. Hurmerinta: Tissue Interactions in Tooth Devalopment

this time cuspal morphogenesis is still advancing. Cell differentiation starts from the area of cuspal tips and proceeds in a cervical direction; the developmental stage remains more advanced in the cuspal areas throughout subsequent development. The morphological differentiation of the mesenchymal cells precedes that of the epithelial cells. The mesenchymal cells directly underlining the enamel epithelium become postmitotic, polarize, and start secretion of predentin, the organic matrix of dentin (Fig. 2C and D). The cells of the enamel epithelium become postmitotic shortly after the mesenchymal cells, but they polarize only after a layer of predentin has been secreted by the odontoblasts (Fig. 2E). The onset of enamel secretion by the ameloblasts (Fig. 2F) occurs at the time when initial mineralization is seen in dentin [65]. Dentin and enamel matrices contain tooth-specific proteins that can be used as markers for odontoblast and ameloblast cell differentiation. Dentin phosphoproteins are the principal noncollagenous proteins in mineralizing dentin [6, 1061. Enamel proteins (also called enamelins or amelogenins) are unique proteins that are found only in enamel matrix [12, 79, 85, 891. Antibodies have been prepared against enamel proteins [20,77], and they may prove useful as probes for studies on ameloblast differentiation. The tissue interactions that control odontoblast and ameloblast cell differentiation have been studied in numerous transplantation and in vitro studies [19, 28, 38, 41, 81, 82, 901. Although the stage of development that can be experimentally achieved greatly depends on the culture method used, no culture conditions have been shown to support cell differentiation in isolated mesenchyme or epithelium: odontoblasts do not differentiate in the absence of epithelium (Fig. 1, arrow 6), and ameloblast differentiation does not take place in the absence of the mesenchymal component. i.e. odontoblasts secreting predentin (Fig. 1, arrows 7 and 8). The latter part of this review will be devoted to a discussion on the suggested mechanisms of tissue interactions controlling cell differentiation.

Mechanisms of Interaction During Cell Differentiation The different mechanisms that have been proposed in the transmission of signals in morphogenetic tissue interactions have been grouped into three main categories: the long-range diffusion of signal sub-

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stances, matrix-mediated interaction, and cell-mediated interaction [23, 73, 751. Long-range diffusion of signal substances is involved in the neuralization of the amphibian gastrula ectoderm [71, 100, 1011, and in the formation of the lens [34,541. The first suggestion that the ECM exerts important functions on cell differentiation was made by Grobstein [22]. Since then various matrix molecules such as collagen and glycosaminoglycans (GAG) have been shown to induce or stimulate cell differentiation during morphogenetic tissue interactions [3, 4, 24, 25, 45, 471. Studies on kidney tubule induction have provided evidence that morphogenetic signals may be mediated by direct contacts between the interacting cells [74]. The studies on tooth development have suggested that the transmission of signals during odontoblast and ameloblast differentiation is either matrix-mediated or cell-mediated. Different approaches have been used in studies on the mechanism of morphogenetic tissue interactions. Based on direct observations on changes at the tissue interface during the inductive period, hypotheses have been presented which have then been tested in experimental studies. These studies have involved interference with the transmission of signals by physical or chemical means [74, 751. The morphogenetic effects of suggested inductive molecules have also been tested on isolated target tissues. Such approaches have also been applied in studies on the mechanisms of tissue interaction during cell differentiation in the developing tooth, and the evidence favoring different types of transmission mechanisms is reviewed below.

Changes at the Epitheliomesenchymal Interface In the early bell stage tooth germ (Fig. 2B), a basement membrane separates undifferentiated enamel epithelial cells from the mesenchymal cells of the dental papilla. A continuous basal lamina is seen lining the epithelial cells, and at its mesenchymal aspect, fibrillar material is oriented perpendicularly to the lamina (Fig. 4A and B). Alignment of mesenchymal cells under the enamel epithelium and their subsequent differentiation into odontoblasts are associated with an increased number of these fibrils [46,88]. Immunofluorescent localization studies have shown that at this time the dental basement membrane is composed of molecules common to basement membranes in general. These include type IV collagen, laminin, a heparan sulfate proteoglycan, and fibronectin [7, 50, 95, 961 (Fig. 4C). At the time of

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Fig. 4. The epithelio-mesenchymal interface of bell stage mouse tooth germs at the time of dontoblast differentiation (Fig. 2B). A In a transmission electronmicrograph, mesenchymal cells are seen to make close contacts with the basal lamina (arrow) under the enamel

epithelium. B A scanning electronmicrograph showing alignment of mesenchymal cells along the network of the basement membrane (arrow). C Immunofluorescence localization of fibronectin. Fluorescence is seen in the mesenchymal tissue and in the basement membranes. The dental basement membrane (arrow) is intensely stained. D An autoradiograph illustrating high incorporation of [%I sulfate into the epithelio-mesenchymal interface (arrow). Abbreviations: M mesenchymal cells, E epithelial cells

mesenchymal cell alignment, the amount of fibronectin is increased, while the amount of the other three components remains unchanged [96]. It has been suggested that fibronectin plays a role in mesenchymal cell differentiation into odontoblasts,

presumably by promoting mesenchymal cell attachment to the basal lamina [95, 96, 981. Using autoradiography we have observed that incorporation of [3H] fucose and [35S] sulfate into the dental basement membrane is also increased at the

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Fig. 5. The epitheho-mesenchymalinterface of bell stage mouse tooth germs at the time of ameloblast differentiation (Fig. 2E). A Immunofluorescent localizationof laminin, a basement membrane glycoprotein.The basement membrane (arrow) is stainedin the cervical and intercuspal areas, but no fluorescence is seen under the polarized epithelial cells in the cuspal area. B A transmission electronmicrograph illustrating the penetration of epithelial cell processes through the basal lamina into predentin.The onset of predentin mineralization is seen. Abbreviations: PD predentin, E epithelial cells

time of mesenchymal cell differentiation (Fig. 4D, and unpublished observations). This suggests that increased synthesis of glycoproteins and GAGS is associated with odontoblast differentiation. The epithelial preameloblasts begin to rearrange their intracellular organelles soon after a thin layer of predentin has been secreted by the odontoblasts [35] (Fig. 2D). Because of this temporal relationship, different components of predentin have been suggested to be involved in ameloblast polarization, and support for these hypotheses has come from studies in which production of predentin has been interfered with (see below). Simultaneously with the onset of predentin mineralization, the basal lamina of the preameloblasts becomes discontinuous and disappears. This has been demonstrated in numerous electronmicroscopical studies [31, 52, 611, and by immunofluorescent localization of basement membrane components [95,96] (Fig. 5 ) . The disappearance of the basal lamina allows the formation of cell-cell contacts between the preameloblasts and the odontoblasts, and these have been proposed to mediate an inductive signal for ameloblast differentiation [31-33,52,84]. Degradation of the basal lamina has been suggested to occur as a result of mesenchymal cell activity. Collagenase activity was demonstrated in coated vesicles appearing in the predentin matrix at the time of basal lamina degradation [87]. These vesicles were earlier shown to contain RNA, which

was suggested to carry morphogenetic information from the mesenchymal to the epithelial cells [81,83]. Because the appearance of these vesicles coincides with the onset of dentin mineralization, the vesicles have generally been associated with the mineralization process [16, 801. Experimental Interference with lnteractions

The transfiZter technique, involving separation of the interacting tissue components and their culture with interposed filters, was originally designed by Grobstein [21]. It has provided an appropriate method for studies on transmission mechanisms [74,105]. The method was first applied to tooth development by Koch [38] who demonstrated that odontoblasts and ameloblasts differentiated in explants in which dental mesenchyme and epithelium from bell stage tooth germs were cultured on different sides of a Millipore filter. More recently we have made use of Nuclepore filters in studies on tooth differentiation [93, 941. These filters have straight pores allowing detailed studies on cytoplasmic penetration into the filter pores [49, 1051. For transfilter culture the epithelial and mesenchymal components of bell stage tooth germs (Fig. 2B) were enzymically separated and cultured on opposite sides of Nuclepore filters with varying pore sizes. Filters with a pore diameter 0.2-0.6 pm allowed differentiation of odontoblasts, first seen as

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Fig. 6. Micrographs of transfilter explants of dental mesenchyme (above) and epithelium, separated from early bell stage tooth germs (Fig. 2B) and cultured with interposed Nuclepore filters.A A light micrograph of an explant cultivated 14 days with 0.2 pm pore-size filter. Differentiated odontoblasts secreting predentin are seen on the upper filter surface and polarized ameloblasts on the opposite side. B A light micrograph of an explant cultivated 3 days with 0.6 TI pore-size fiiter. Mesenchymal cells have aligned on the upper side of the filter, which was considered as the first sign of odontoblast differentiation. C A transmission electronmicrograph of the lower filter surface of an explant like 6B. A continuous basal lamina with associated fibrillar material is seen between the epithelial cells and the filter. A mesenchymal cell process that has traversed the filter is seen in close association with the basal lamina (arrow). Abbreviations: M mesenchymal cells, 0 odontoblasts, PD predentin, E epithelial cells, A ameloblasts

alignment of cells on the filter surface (Fig. 6B), and later as secretion of predentin (Fig. 6A). This was followed by differentiation of ameloblasts and enamel secretion on the opposite side of the filter [93]. As seen in ultrastructural examination, these filters also allowed penetration of cell processes into the filter pores. At the time of preodontoblast alignment, mesenchymal cell processes had traversed the filter and were seen in close association with the basement membrane between the epithelial cells and the filter [94] (Fig. 6B and C). Differentiation was prevented by filters with O.l-pm pore size, which also were

shown to inhibit ingrowth of cell processes into the filter pores. These filters have been shown to allow rapid diffusion of large molecules and even viruses [%I. Results of our transfiiter experiments suggested that differentiation of the mesenchymal cells into odontoblasts was triggered by close contacts between the differentiating cells and the basement membrane, and not by diffusible molecules from the epithelium. The mechanism of ameloblast differentiation could not be studied because the small pore size filters blocked the chain of interactive events already at the stage of odontoblast differentiation.

I. Thesleff and K. Hurmerinta: Tissue Interactions in Tooth Development

Fig. 7. A photomicrographillustrating the effect of diazo-oxo-norleucine (DON) on mesenchymal cell differentiation.An early bell stage mouse tooth germ was first cultured 2days in control medium; by then the cuspal odontoblasts had differentiated (not shown). The explant was then transferred to medium with DON (50w) and subcultured 7days. A boundary (arrow) is seen between the undifferentiated mesenchymal cells and odontoblasts secreting predentin. DON thus has prevented the differentiationof mesenchymal cells, but it has not inhibited later differentiative events such as predentin secretion by already differentiated odontoblasts and ameloblast polarization. Abbreviations: M mesenchymal cells, 0 odontoblasts, E epithelial cells

Cell differentiation during tooth development can be inhibited by interfering with various cellular functions by a number of chemiculugena [l,2,10,11, 53, 56, 69,781. The most relevant information about the mechanisms of the epithelio-mesenchymal interactions has come from experiments in which the synthesis of various ECM molecules has been affected. As described above, the ideas of the importance of matrix molecules were prompted by observations on the temporal relationship between ECM changes and cell differentiation. The major ECM molecules in the basement membrane are collagen, proteoglycans, and glycoproteins [36]. Predentin is composed mainly of type I collagen [9], but also of small amounts of GAGs and glycoproteins [27, 861. Interference with collagen deposition by collagenase,L-azetidine carboxylic acid, &minoproprionitrile, tetracycline, or a,a’-dipyridyl prevents both tooth morphogenesis and odontoblast differentiation [14,26, 37, 39, 671. Reversal of the inhibition in some cases by exogenously added procollagen indicates the importance of the collagen molecule itself [15, 371. The actual role of collagen in odontoblast differentiation is, however, difficult to evaluate, since these chemical agents also prevent the

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expression of odontoblast differentiation, i.e. , the deposition of the collagenous predentin. Odontoblast differentiation can be prevented by inhibition of glycoconjugate synthesis. Inhibition of differentiation by diazosxo-norleucine (DON) (Fig. 7) and vitamin A is accompanied by decreased [35S]sulfate incorporation [29, 301 (Fig. 8A and B). Inhibition of protein glycosylation by tunicamycin also prevents odontoblast differentiation [97, 981. In tooth germs exposed to vitamin A and tunicamycin, the deposition of the basal lamina was impaired (Fig. 8C and D). Furthermore, tunicamycin decreased significantlythe amount of fibronectin in the basement membrane region [98]. These results were interpreted to suggest that GAGs and glycoproteins have important functions in the proposed cell-matrix interaction involved in odontoblast differentiation. DON, vitamin A, and tunicamycin do not prevent deposition of predentin or differentiation of ameloblasts, if these agents are added after initiation of predentin secretion [29,30,97,98]. This suggests that GAGSand glycoproteins in the predentin matrix are not required for polarization of the ameloblasts and onset of enamel secretion. Current Hypotheses It is evident that the differentiation of the dental mesenchymal cells into odontoblasts is controlled by the enamel epithelium, and that this epithelio-mesenchymal interaction is of a permissive character. In other words, the dental mesenchyme has been ‘predetermined’ during earlier interactions, and the role of the epithelial tissue is merely to create favorable conditions under which the new phenotypic character of the mesenchymal cells can be expressed. On the basis of the descriptive and experimental studies reviewed above, it seems justified to assume that the basement membrane has an important role in this function of the enamel epithelium (Fig. 9). The major components of the basement membranes are of epithelial origin [36]. It was recently shown that [3H]glucosamine-labelledmaterial accumulating into the dental basement membrane is synthetized by the epithelium [13] and also that isolated enamel epithelium is able to deposit a normal-appearing basal lamina [a]. Various ECM materials such as collagen gels or epithelial secretory products, e.g., lens capsules do not, however, stimulate odontoblast differentiation in isolated dental mesenchyme [a, 921. Experiments in which tooth germs were exposed to bromodeoxyuridine (BrdU) have suggested that

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Fig. 8. Effects of vitamin A and tunicamycin on the epithelio-mesenchymalinterface of cultured tooth germs. Both agents inhibited the differentiation of odontoblasts. A An autoradiograph demonstrating the incorporation of [%I sulfate after 4 days of culture in control medium. High incorporation is seen at the epithelio-mesenchymalinterface (arrow). B In a similar explant cultured in the presence of vitamin A (4 pg/ml) much less incorporation of [3sS]sulfate is seen. C An electronmicrograph of separated and subsequently recombined epithelial and mesenchymal components of tooth germs. After 2 days of culture in control medium, a continuous basal lamina (arrow) has been restored. D The basal lamina (arrow) restored in the presence of tunicamycin (0.15 pg/ml) is not continuous, and less fibrillar material is seen at its mesenchymal aspect. Abbreviations: M mesenchymal cells, E epithelial cells

DENTAL MESENCHYME -0DONTOBLASTS Fig. 9. Schematic presentation of the suggested mechanism of the epitheliomesenchymal interaction involved in odontoblast differentiation. A cell-matrix type of interaction between the basement membrane and the mesenchymal cells may trigger odontoblast differentiation

undisturbed epithelial cell activity is needed for odontoblast differentiation. BrdU treatment of the epithelial component caused inhibition of odontoblast differentiation, whereas treated mesenchymal cells were still able to differentiate [69]. What then is the mechanism by which the basement membrane controls odontoblast differentiation? One possibility is that passage of some inductive material from the epithelial to the mesenchymal cells occurs by endo- and exocytosis via the basal lamina, as has been suggested for vaginal epithelial differentiation [8]. More consistent with the available data, however, is the possibility that a cell-matrix type of interaction takes place between

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I. Thesleff and K. Hurmerinta: Tissue Interactions in Tooth Development

ENAMEL EPITHELIUM

MELOBLASTS

Fig.10. Schematic presentation of the suggested mechanisms of tissue interaction in ameloblast differentiation. An interaction between the differentiating cells and the predentin matrix may be important. Also heterotypic cell-cell contacts between the odontoblasts and the epithelial cells, seen after disruption of the basal lamina, may play a role in ameloblast differentiation

the basement membrane and the mesenchymal cell surface. The fibrous lattice of the basement membrane is evidently created as a result of interactions of various macromolecules. GAGs are known to bind to collagen [51] and fibronectin can bind to different types of collagens and to certain GAGs [64,104,107]. In fact, fibronectin may, as was recently suggested, serve as a cross-linking structural glycoprotein in the basement membranes, and may play an important role in their internal organization [108]. Furthermore, all the major macromolecules interact with the plasma membrane, e.g., fibronectin seems to have a role in promoting cell attachment to collagen [81, 1041. The existence of these interactions between the macromolecules and the cell surfaces can explain the observations that interference with the synthesis of any of these molecules inhibits differentiation of odontoblasts. In these experiments the organization of the basement membrane and/or the cell periphery was presumably disturbed, resulting in failure of the cell-matrix interaction. Differentiation of the enamel epithelial cells into ameloblasts can be regarded as the terminal step in the progressive determination of the epithelial tissue (Fig. 1). As discussed above, mesenchymal tissue is required for ameloblast differentiation to take place. This dental mesenchyme is capable of inducing ameloblast differentiation also in nondental epithelia, and the indication is that the mesenchymal influence on epithelial differentiation is directive in nature. However, when heterotopic epithelia are cultured with dental mesenchyme, the epithelium always

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undergoes typical enamel organ morphogenesis prior to overt differentiation of ameloblasts, which is seen only after the odontoblasts have initiated predentin secretion. As discussed in the beginning of this review, this later epitheliomesenchymal interaction may be permissive: the epithelium has already reached a high level of differentiation, and the number of options for its differentiation has been limited. It seems that predentin collagen is one factor controlling the polarization of ameloblasts (Fig. 10). Glycoproteins and sulfated GAGs, also present in predentin, do not appear to be necessary for ameloblast polarization. Other features that have been associated with ameloblast polarization and enamel secretion are the breakdown of the basal lamina and formation of heterotypic cell-cell contacts between the odontoblasts and the preameloblasts (Fig. 10). These events may have functions, e.g., in the onset of the secretory activity of the ameloblasts, although so far there is no experimental evidence to support these hypotheses.

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Received June 1980/Accepted November 1980