Epithelial-mesenchymal interactions during odontogenesis II. Intercellular matrix vesicles

Mechanisms of Ageing and Development

Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

E P I T H E L I A L - M E S E N C H Y M A L I N T E R A C T I O N S D U R I N G ODONTOGENESIS II. I N T E R C E L L U L A R MATRIX VESICLES

HAROLD C. SLAVKIN, PABLO BRINGAS, Jr., RICHARD CROISSANT and LUCIEN A. BAVETTA Department of Biochemistry, School of Dentistry and the Graduate Program in Cellular and Molecular Biology, University of Southern California, Los Angeles, California (U.S.A.)

(Received February 2nd, 1972)

SUMMARY The present study has demonstrated morphologically the probable sequential formation of matrix vesicles during embryonic rabbit incisor tooth formation. The observations presented suggest that these vesicles may have been selectively formed within each cell type and then exported into the matrix, or that matrix vesicles may have been formed by a pinocytotic mechanism. It is uncertain on the basis of our morphological data whether one or both of these explanations is adequate. Matrix vesicles were increasingly more concentrated in the matrix region adjacent to dividing inner enamel epithelia and preodontoblast mesenchyme. Thereafter, the concentration of matrix vesicles diminished with the cessation of cell division within each cell type. The vesicles varied considerably in size (500 A to 0.1 /~m), shape and the electron density of their contents. They were limited by a unit trilaminar membrane, often coated with a filamentous mat, and contained materials of varying electron density. Morphological data indicate that vesicles observed in the developmentally more advanced dentine organic matrix (in association with nondividing cells) function in the initiation of calcification during dentinogenesis. On the basis of morphological information we anticipate that additional developmental events within dissimilar cell types and within the forming organic matrix may be mediated through matrix vesicles. The interpretation of vesicle functions on the basis of matrix vesicle ultrastructure and location with respect to each cell type must be highly qualified until additional criteria can be employed. However, with regard to possible informational and structural macromolecular transmission and/or ion transport by matrix vesicles, the morphological evidence presented may be highly significant.

INTRODUCTION Somatic cells within metazoan organisms contain all the genetic information required for cell differentiation as expressed through biochemical and cytological Mech. Age. Dev., 1 (1972) 139-161

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processes. It has been repeatedly and convincingly demonstrated, however, especially during embryogenesis, that cell differentiation can not be initiated in eucaryotic cells without exogenous, epigenetic stimuli. One method of studying this phenomenon has been to observe embryonic inductive tissue interactions between epithelium and mesenchyme (see extensive reviews) 1-4. Epithelial-mesenchymal interactive processes have repeatedly been shown to be one of the basic mechanisms controlling morphogenesis. In that morphogenetic interactions between these heterotypic tissues do not require intercellular contacts between interactants, transmissible factors have been sought for the embryonic inductive effects1,5, 6. Numerous studies unequivocally show that direct mesenchymal cell contact with the adjacent epithelium is not a requirement for embryonic induction and, therefore, research efforts have recently been directed towards an understanding of extracellular materials during embryogenesis (see reviews) 1,4,7. Despite suggestive experimental results, the mechanism for the transmission of developmental information between heterotypic tissues and the nature of the epigenetic stimuli remain unknown4,6, 8. One suggested source for information with which to confront this major problem in developmental biology is the characterization of "extracellular matrix constituents" interposed between tissue interactants in situ or in vitro 7,9,1°. Newly synthesized ribonucleic acid (RNA) has been identified as a component of the intercellular matrix interposed between epithelium and mesenchyme during odontogenesis in the embryonic rabbit~L Autoradiographic studies using light microscopy showed that, when tooth germs were incubated with a variety oftritiated nucleosides, label was transferred from both cell types into the matrix. The radioactivity observed over the matrix could be removed by treatment with ribonuclease and its intracellular passage and subsequent export was shown to be inhibited by actinomycin-D. Recently in this laboratory, several methylated RNA's of low molecular weight were isolated from the intercellular matrix of developing embryonic teeth lz. Recent studies indicate that isolated intercellular matrices devoid of adherent cells enhance cytodifferentiation within either epithelium or mesenchyme in vitro lz. In each of these studies, ultrastructural observations indicated that membrane-bound, electron-dense bodies were present within the isolated matrices prior to phenol extraction; these structures were not found following extraction procedures. Such indirect data stimulated us to examine more closely the developing intercellular organic matrix in situ for evidence of the formation of the membrane-bound, electron-dense bodies. Recently, it has been well-established that extracellular membrane-bound structures are found in organic matrices which calcify 14. These extracellular structures, termed matrix vesicles15, have been found to contain proteolytic enzymes 16 or structural organic matrix constituents 17 associated with cartilage formation. Matrix vesicles within cartilage matrix formation14-~6,18, a9 have been implicated as a means for local concentration of calcium and phosphate ions necessary for calcification. Although numerous investigators have studied general ultrastructural aspects of odontogenesis 2°-28, previous studies have not reported matrix vesicles as actual constituents of the intercellular organic matrix. This paper reports the presence of matrix vesicles in the intercellular organic 140

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matrix interposed between inner enamel epithelium and adjacent preodontoblasts throughout the germinative region of embryonic rabbit incisor tooth primordia. Such matrix vesicles are observed to be a characteristic feature of' odontogenesis in this region and repeatedly are found after a variety of different fixation methods which eliminate or reduce the possibility of membrane vesiculation due to tissue preparation. The presence of matrix vesicles within the extracellular milieu between heterotypic tissues may bear upon a number of developmental problems. Therefore, a morphological description appears most appropriate at this time. MATERIALS AND METHODS New Zealand white rabbits, 25-days pregnant, were sacrificed by injecting 10 ml of air into the left ear vein. The germinative region of maxillary and mandibular incisor tooth primordium was employed for all morphological observations. This region of the incisor tooth primordia demonstrates an increasing gradient of progressive cytodifferentiation within both epithelium and adjacent mesenchymal cell populations as well as an increasing gradient of intercellular organic matrix formation. Each embryonic tooth provides, therefore, a continuous opportunity to observe numerous stages of development in two embryologically dissimilar cell populations and their respective organic matrices. At the stage of organ development selected in these studies, representative aspects of all types of tooth formation are evident, e.g. cell differentiation, specific extracellular matrix protein synthesis, mineralization and calcification. All observations were limited to the germinative or cervical loop regions of incisor tooth primordia to avoid the complications which would be introduced by the process of amelogenesis. Tooth primordia for microscopy were removed from embryonic rabbits randomly selected from nine litters of animals. Procedures for fixation, dehydration, embedding, sectioning and staining were essentially those reported by Hay and Revel ~9. Primary fixation was for 20 min in a formaldehyde-glutaraldehyde fixative30 with cacodylate buffer at pH 7.2. After thoroughly rinsing in buffer at 4 °C, tissues were postfixed in osmium tetroxide, buffered to pH 7.2, for 60 min at 4 °C. Specimens were then rinsed in 0.1 M maleate buffer at pH 5.0 for 5 rain and stained en bloc for 60 min in a 2 ~ solution of uranyl acetate in 0.1 M maleate buffer (pH 5.0). The tissues were rinsed in the maleate buffer and dehydrated rapidly through a graded series of alcohols and propylene oxide. Replicate tooth specimens were processed similarly except they were not stained with uranyl acetate. Tissues were embedded in Epon 812 (ref. 31) and polymerized at 60 °C for 3648 h. Excised tooth primordia were also fixed and dehydrated by another method previously employed in our laboratory 2°. Sections 0.5-1 micron thick were cut and examined directly by phasecontrast microscopy, or by light microscopy after staining with 1 ~o toluidine blue, in order to obtain the desired orientation of each block. In addition, isolated intercellular organic matrices from 25-day embryonic rabbit incisor teeth were prepared for electron microscopic observations by methods previously reportedlL Observations were made on isolated, sonicated intercellular matrices devoid of adherent cells. Mech. Age. Dev., 1 (1972) 139-161

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Sections with silver to pale gold interference colors were cut with a diamond knife on a Sorvall MT-2 ultrarnicrotome and observed either unstained or stained with lead citrate 32 for examination of fine-structure. Serial sections with pale gold interference color, were cut to assess the actual dimensions of the extracellular matrix vesicles. Electron microscopy was done using a Zeiss EM-9S instrument. RESULTS

General description Along the labial aspects of the developing incisor tooth primordia in rodents and rabbits there is an evident, continuous gradient of increasing cytodifferentiation within each of the heterotypic cell populations contributing to odontogenesis (Fig. 1). Those cells adjacent to the basement membrane seen in the light microscope show a cellular architecture illustrative of merocrine-like secretory cells actively engaged in the synthesis and export of protein. The intercellular organic matrix interposed between these heterotypic cells is formed as a result of continuous, reciprocal epithelial-mesenchymal interactions. In the germinative regions of the developing tooth primordia the intercellular organic matrix forms concomitantly with increasing cytodifferentiation, each cell synthesizing and exporting a tissue-specific protein. Along the increasing gradient of matrix formation, first of dentine and then of enamel, the epithelial cells cease to divide at the level at which the organic matrix is 15-20 #m thick. The reader is referred to several recent reviews of odontogenesis 33-34, investigations of detailed aspects of the ultrastructure of the forming intercellular matrix during embryonic rabbit incisor development 2°, and the fine-structure of secretory ameloblasts21,~-5,2s, 35 and odontoblasts22, z3 at various stages of development during mammalian tooth formation. Observations of matrix vesicles in progenitor dentine The observations to be reported are limited to the germinative regions of the developing incisor tooth in the embryonic rabbit during the 25th day of gestation. These observations are limited to the distal or secretory poles of the inner enamel

Fig. 1. Epon-embedded specimen of undecalcified 25-day embryonic rabbit incisor tooth primordium demonstrating the germinative region. Note the increasing gradient of cytodifferentiation within epithelia (outer enamel epithelium, OEE; inner enamel epithelium, IEE) and adjacent mesenchyme (preodontoblasts, PO; odontoblasts, O). Note the significant thickening in the characteristic basement membrane (BM) interposed between heterotypic cells becoming the intercellular organic matrix (IM). The asterisk (*) indicates a region comparable to that shown in Fig. 2. × 266. Fig. 2. Surveyelectron micrograph of the interface between epithelium and adjacent preodontoblasts. The secretory regions of both cell types contain a variety of membranous organelles, e.g. mitocbondria, rough endoplasmic reticulum, coated vesicles, and numerous arrays of polysomes in proximity to the basal lamina (BL). In this region no matrix vesicles wele observed. Note the numerous cell processes extending from the mesenchymal cells and the collagen fibrils dispersed throughout the adjacent ground substance. × 6100. 142

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epithelia and adjacent preodontoblast cell populations. These epithelial and mesenchymal cells, the prospective ameloblasts and odontoblasts, are characterized by various membranous organelles including many mitochondria, lysosome-like bodies, multivesicular bodies, secretory vesicles, Golgi bodies and numerous arrays of rough endoplasmic reticulum. Within both heterotypic cell populations numerous free ribosomes were observed. An overt characteristic of the mesenchymal cells is the numerous cell processes extending towards the undersurface of the basal lamina in association with the adjacent inner enamel epithelium (Fig. 3). Direct cell contacts between mesenchymal cells and epithelial cells were not observed. Within the intercellular organic matrix interposed between these dissimilar cell populations, numerous collagen fibrils (predentine collagen) were noted in longitudinal and in cross-section, and an accumulation of microfilaments were adherent to the undersurface of the inner enamel epithelial cells (Figs. 2-4). Descriptions of secretory vesicles within ameloblasts25, ~s and odontoblasts ~a, and excellent accounts of their locations within the cytoplasm, have been reported. During our studies, a variety of vesicles of varying size and shape were noted within the cytoplasm of both inner enamel epithelium and preodontoblasts, in close association with the plasma membranes of both cell types, the basal lamina and as actual (arrows) components of the intercellular organic matrix (Fig. 3). Regardless of fixation or dehydration procedures we repeatedly observed membrane-limited profiles in association with preodontoblast plasma membranes, the basal lamina and in the intercellular organic matrix (matrix vesicles). Within the secretory poles of both inner enamel epithelium and preodontoblasts, and in the intervening organic matrix, concentrations of numerous circular and oval profiles measuring 500 A to 0.1/zm in diameter were observed (Figs. 3 and 4). The concentration of vesicles within each cell type and within the adjacent intercellular matrix increased proportionally with increases in cytodifferentiation within the inner enamel epithelial (-+ameloblasts) and immediately adjacent mesenchymal cells (-~preodontoblasts+odontoblasts). Intracytoplasmic and extracellular matrix vesicles were often bounded by a limiting membrane (75-150 A thick) of the trilaminar unit membrane type (Figs. 4-6). Many of the intracytoplasmic and matrix vesicles were coated (Figs. 6-11). Large multivesicular bodies were repeatedly observed within the secretory poles of both epithelial and preodontoblast cells.

Fig. 3. A survey electron microgiaph of a more advanced region of forming matrix (progenitor mantle dentine) interposed between inner enamel epithelia (lEE) and adjacent preodontoblasts (PO). At this level of development the matrix is about 3/~m thick. The preodontoblasts have become low columnar with an abundant infranuclear region filled with rough endoplasmic reticulum, polysomes and numerous coated and uncoated secretory vesicles. Numerous mesenchymal cell processes (CP) are observed as constituents of the forming organic matrix, some of which extend in close proximity to the basal lamina (asterisk (*) and upper rectangle indicating area shown in Fig. 4). The nuclear polarity is not as yet overt within the epithelial cells at this level of development. Note the numerous extracellular vesicles (arrows) in close proximity to the basal lamina (BL) within the organic matrix. × 9000. Insert: an enlargement of the lower rectangle. Matrix vesicles (MV) are easily discerned from cell processes. × 25 500. Mech. Age. Dev., 1 (1972) 139-161

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The incidence of intercellular matrix vesicles is striking. In the more germinatire regions of Hertwig's epithelial root sheath (Fig. 1, asterisk*), there are no matrix vesicles (Fig. 2). As the less-differentiated mesenchymal cells adjacent to the forming organic matrix form intercellular contacts, they acquire characteristics associated with merocrine-type preodontoblasts. It is in this region that matrix vesicles first were observed (Fig. 3). As the inner enamel epithelia and adjacent preodontoblasts become polarized, there is an appreciable thickening (approx. 3 #m) of the intercellular organic matrix (Fig. 12). In this region there is an appreciable concentration of discrete matrix vesicles (Figs. 4 and 5). The presence of preodontoblast cell processes within the forming intercellular organic matrix made it necessary to distinguish between cell processes and matrix vesicles (Figs. 3 and 4). Serial sectioning (silver) and screening of large numbers of specimens from nine different litters of animals enabled discrimination between cell processes from the preodontoblasts, odontoblasts, cell "debris" and matrix vesicles. In serial sections the matrix vesicles (500/~ to 0.1 #m) disappear, whereas the cell processes can easily be followed. The granular, electron-dense material(s) within matrix vesicles varied considerably with respect to size distribution of granules within vesicles (Figs. 4-6). The granules within many of the larger vesicles (approx. 0.1/~m) were comparable in size to ribosomes (Figs. 5, 7, 9 and 11). On the basis of size and the electron density of their contents, three morphologically different types of matrix vesicles were recognized within the forming organic matrix: small with electron-dense granular contents (MV1); larger with electron-dense granular contents (MV2); and larger with discrete, ribosome-like granules as contents (MV3). Each type was found to be easily distinguished from cell processes (Figs. 4-7). All three types were observed in the intercellular organic matrix region adjacent to rapidly dividing inner enamel epithelial and preodontoblast cells (Fig. 12). Serial sections through numerous specimens also indicated that intracytoplasmic secretory vesicles were to be found in various relationships with plasma membranes of both cell types, including being outside of epithelial and mesenchymal cells (Figs. 4-11). Many of the extracellular matrix vesicles were comparable to those secretory vesicles within epithelial and mesenchymal cells. The physical measurements (500 A to 0.1/~m diameter) of the matrix vesicles were similar to those of the hetero-

Fig. 4. Higher magnification electron micrograph at the level of development illustrated in Fig. 3 (asterisk, *) upper rectangle which indicates several cell processes (CP) which closely approximate the undersurface of the epithelium (IEE). Matrix vesicles of varying sizes, shapes and electron densities are easily discriminated from collagen fibrils (C) in longitudinal, transverse or cross-sections. In this section two types of matrix vesicles are indicated (MVI and MV~). The apical regions of epithelium contain rough endoplasmic reticulum (RER) and numerous vesicles,often fusing with the plasma membrane and expelling their contents into the maUix regions (arrows). Mesenchymal cell processes contain similar coated secretory vesicles apparently emptying their contents farrows) into the forming dentine organic matrix. × 48 000. Mech. Age. Dev., 1 (1972) 139-161

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Fig. 5. The infranuclear region of inner enamel epithelium at a more advanced stage of differentiation which contains numerous membranous organelles, e.g. mitochondria (M), rough endoplasmic reticulum (RER), and coated secretory vesicles (CSV). Along the undersurface of the epithelium numerous matrix vesicles were noted (MV1, MV2 and MV3) as well as preodontoblast cell processes (CP). N, nucleus. × 26 400.

geneous population of secretory vesicles within the cells. The same degree of variation in size and electron densities was noted (Figs. 4-6). The spatial orientation of intracytoplasmic secretory vesicles with respect to the plasma membrane of each cell type showed appreciable variations. Intracytoplasmic vesicles were seen to fuse with the plasma membrane (Figs. 4 and 10) or appeared to "bud" from the cell membranes (Figs. 4, 7 and 12). In regions in which intracytoplasmic vesicles approximated the adjacent inner surface of the plasma 148

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Fig. 6. Within the forming matrix prior to the degradation of the basal lamina (BL) numerous vesicles are actual constituents of the forming mantle dentine. Serial sections through this region indicate that some of the membrane-limited structures are cell processes. Collagen (C) is easily identified. The asterisk (*) indicates a region containing three matrix vesicles (insert). Insert: three vesicles limited by a trilaminar unit membrane (arrows) and containing electron-dense, granular material (MV3). × 37 100. Insert × 85 100.

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membrane, we observed an area of higher electron density at this interface (Figs. 4, 8, 10 and 12). Most of the intracytoplasmic secretory vesicles and those observed in the organic matrix (matrix vesicles) were coated with a filamentous mat (Figs. 4 and 6). In some instances the source of the vesicles appeared to be multivesicular bodies within preodontoblast cell processes (Fig. 12). The concentration of matrix vesicles increased within the forming organic matrix to the level associated with the cessation of cell division within the inner enamel epithelia (Figs. 13 and 15). Throughout this region of the developing tooth primordium, from the cervical tip of Hertwig's epithelial root sheath (Fig. 1)to the level of nondividing ameloblasts (Fig. 13), the distribution of matrix vesicles appeared heterogeneous; size, shape and electron densities of the material(s) within the vesicles varied considerably. In the predentine organic matrix (Fig. 13), in association with the nondividing cell types, the vesicles appear fewer in number and more homogeneous (Figs. 15 and 16). Closer examination of the matrix vesicles in this matrix region, specifically those close to the odontoblasts, indicates that these vesicles are associated with the initial stages of calcification (Fig. 16). In this region the vesicles appear to contain electrondense, granular material closely associated with the odontoblast cell processes. As the predentine organic matrix thickens, there are numerous "nucleation" areas of calcification close to the matrix vesicles and the fibrous components of the forming organic matrix (Fig. 16).

Matrix vesicles and their relationship to calcification Specimens examined in this investigation were not decalcified. The selection of the germinative region of the tooth primordium provided an opportunity to observe continuously the developing intercellular organic matrix (progenitor mantle dentine) from the appearance (Fig. 1) of a thickened basement membrane (0.5 #m thick) to the formation of a discrete organic matrix (20 #m thick), termed progenitor mantle dentine (Figs. 12 and 13). Light microscopic observations included the entire

Fig. 7. Microvilli or cell processes extend from the epithelium following degradation of the basal lamina (arrows). Discrete matrix vesicles (MVs) are seen in close proximity to the inner enamel flEE) epithelial outer cell surface. The matrix vesicles contain electron-dense granules comparable in size and density to ribosomes (R) observed throughout the cytoplasm. Note the coated vesicles ¢CV) within the epithelium, x 75 200. Fig. 8. Multivesicular bodies (MVB) and coated vesicles (CV) in proximity to the plasma membrane of a preodontoblast cell process (CP). × 85 200. Fig. 9. Matrix vesicles (MV3) limited by a trilaminar unit membrane (arrows) containing ribosomelike, electron-dense granules. Note the proximity to a preodontoblast cell process (CP). x 128 500. Fig. 10. Within the forming epithelial Tomes' processes rough endoplasmic reticulum (RER) and clusters of ribosomes were observed. Indications of different stages of merocrine-typesecretion were evident (arrows). Note the matrix vesicle containing granules enclosed within another unit membrane structure, x 87400. Fig. 11. Many of the matrix vesicles (MVs) appear to "pinch-off" from preodontoblast cell processes (CP). Note the numerous ribosome-like granules within the vesicle (arrows). × 87400.

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germinative region (Hertwig's epithelial root sheath area) and the earliest zones of calcification (Fig. 13, arrow). Prior to the initiation of calcification within less-developed progenitor mantle dentine (Fig. 12), numerous matrix vesicles were noted close to the basal lamina and close to the odontoblast secretory poles (Fig. 14, arrows). At a higher level of matrix formation (Fig. 13), matrix vesicles (approx. 0.1 # m in diameter) were in association with small clusters of "needle-like" material (Fig. 16). In this region of matrix tormation the basal lamina, previously associated with the undersurface of the inner enamel epithelia (Fig. 14), was not evident. The secretory portions of the preameloblast cells (inner enamel epithelium) appeared as cell projections or microvilli (Fig. 15). The preponderance of matrix vesicles throughout this region of mantle dentine was associated with amorphous, granular, electron-dense material (Fig. 16, arrows). Large collagen fibers (with characteristic 640-A banding) were observed in longitudinal, transverse, and cross-sectional planes. The matrix vesicles were easily distinguished from collagen fibers within the progenitor mantle dentine (Figs. 15 and 16). Isolation o f the intercellular matrix containing vesicles Previously we reported procedures which enabled isolation of the cervical intercellular organic matrix (10-20/zm thick) containing R N A during tooth formation in rabbit embryos 12. During the present investigation we routinely noted the presence of numerous matrix vesicles with diameters of 500/~ to 0.1 #m within the isolated matrix (Fig. 17, arrows). The larger vesicles contained electron-dense granular material and were coated. Matrix vesicles were distributed throughout the matrix even after microdissection and ultrasonication procedures. Previously we reported that the vesicles (membrane-limited, electron-dense bodies) were removed by the phenol extraction procedures used to extract R N A 12. The larger (0.1 # m in diameter) matrix vesicles were randomly distributed in the matrix; however, we observe that most of the vesicles ( < 0.1 #m in diameter) were to be found close to the undersurface of the inner enamel epithelium in situ (Figs. 5 and 6) and after isolation procedures (Fig. 17). This observation persisted throughout our studies; numerous matrix vesicles were repeatedly seen in proximity to that area previously occupied by the epithelium regardless of fixation, dehydration or plane of section.

Fig. 12. Along the gradient of increasing cytodifferentiation and dentine organic matrix formation (IM), both inner enamel epithelium (lEE) and preodontoblast cells (PO) reach a level in which mitotic activity diminishes and the cells become columnar in appearance. Note the increasing nuclear polarity in both cell types in this region. × 1000. Insert: an enlargement of the rectangle. Matrix vesicles (MVa and MV~) appear to be exported from the preodontoblast cell process (CP). × 66 300. Fig. 13. At a higher level of dentine matrix (IM) formation in which cell division within both cell types has ceased, initial indications of calcification were evident (arrow) in Epon-embedded, undecalcified, thick sections stained with 1 ~ toluidine blue. IEE, inner enamel epithelial cells becoming preameloblasts; Od, preodontoblasts becoming odontoblasts. × 1000. (Asterisk (*) indicates a region comparable to that shown in Fig. 15.) Mech. Age. Dev., 1 (1972) 139-161

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Cell processes from either epithelial or mesenchymal cells were not observed in sonicated preparations of isolated matrices (Fig. 17). Vesicles within the matrix material (a significant amount of collagen being present) were evident in a variety of shapes, sizes and electron densities, so that we can not report, at this time, a general pattern for these structures. Infrequently, amorphous, electron-dense, granular material was evident in the absence of a unit-type limiting membrane. The matrix region that was isolated did not show early stages of calcification of dentine. DISCUSSION Several questions can be asked about the significance of the presence of matrix vesicles between epithelial and mesenchymal cells during embryonic tooth formation. First, are both cell types forming and secreting matrix vesicles or might only one cell type be responsible for the appearance of extracellular matrix vesicles? Secondly, in that numerous vesicles were observed within the organic matrix (varying on the basis of size and electron density), are there several biological "functions for these vesicles? Are there different types (MV1, MV2 and MV3) of matrix vesicles? For example, might we be observing the more generalized phenomenon of extracellular matrix formation (dentinogenesis) and, in addition, a more discrete phenomena of intercellular communication between heterotypic cells? It would appear evident from numerous studies in the past (see reviews) 36,av that both cell types are actively making fibrous proteins for the formation of extracellular matrix (the epithelial enamel protein(s) and the mesenchymal dentine tropocollagen). The transport of secretory proteins from membrane-bound or free polysomes to the extracellular space is a major activity in a variety of merocrine-type cells. Many of the intracytoplasmic secretory vesicles contact the plasma membrane of the respective cell type as is commonly seen in merocrine secretory cells. The incidence of extracellular matrix vesicles within the forming organic matrix indicated that the majority of vesicles were to be found next to rapidly dividing inner enamel epithelium and adjacent preodontoblasts (Figs. 6 and 12). All three types of matrix vesicles were present (MVa, MVz and MVa). As the mantle dentine formed and the adjacent cells stopped dividing and differentiated into preameloblasts and odontoblasts (Figs. 13 and 15), matrix vesicles containing ribosome-like granules

Fig. 14. This survey electron micrograph demonstrates the basal lamina (BL) and matrix vesicles (arrows) throughout the forming intercellular matrix (2 I~m thick) comparable to the matrix region shown in Fig. 12. As the mantle dentine matrix thickens the basal lamina degenerates and disappears. M, mitochondria within preodontoblasts. × 10500. Fig. 15. This survey electron micrograph demonstrates the characteristics of the region shown in the center of Fig. 13 (asterisk, *). This is a zone of initial calcification within mantle dentine. Note the odontoblast cell processes (CP) and the many matrix vesicles (arrows). In this region the basal lamina is not present and numerous cell processes characterize the undersurface of the inner enamel epithelium. The small black dots may represent hydroxyapatite crystals. × 10 500. Mech. Age. Dev., 1 (1972) 139-161

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Fig. 16. This electron micrograph shows at higher magnification the appearance of the crystals shown in Fig. 15. At this level of dentine formation (Fig. 13, asterisk), electron-dense granular materials (crystals2) appear randomly distributed in association with matrix vesicles (arrows), odontoblast cell processes (CP) and collagen (C). At this level of development the dentine organic matrix is rich in collagen fibrils and fibers, x 31 400.

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(MV3) were no longer evident in the extracellular matrix region. Thereafter, the MV1 and MVz vesicles appeared to be associated in the regions of initial mineralization and calcification of the forming dentine organic matrix (Figs. 15 and 16). The present study has demonstrated morphologically the probable sequential formation of matrix vesicles during embryonic rabbit incisor tooth formation. The observations presented do suggest that these vesicles may have been selectively formed within each cell type and then exported into the matrix, or that matrix vesicles may have been formed by a pinocytotic mechanism (Figs. 3-7 and 14-16). It is uncertain on the basis of our morphological data whether one or both of these explanations is adequate. The interpretation of vesicle functions on the basis of matrix vesicle ultrastructure and location with respect to each cell type must be highly qualified until additional criteria can be employed. However, with regard to possible macromolecular and/or ion transport by some of the matrix vesicles, the morphological evidence presented may be significant. Ultrastructural observations have indicated dense bodies in the extracellular matrix regions during salivary gland, lung, pancreas, skin, feather and hair development at stages in which epithelial-mesenchymal interactions are assumed to be occurring 7. These dense bodies appear to be 0.1/tm in diameter. The origin, function or composition of these dense bodies is unknown. If one accepts the possibility that matrix vesicles (Figs. 5-12) may be involved in the transmission of embryonic induction, the presence of matrix vesicles within the interface of an epidermal organ rudiment known to be engaged in epithelial-mesenchymal interactions 38-42 may complement this hypothesis and provide additional information with which to interpret previously reported data. Recent analyses of transfilter induction experiments suggest that the simple diffusion of "informational macromolecules" is not a comprehensive explanation for the transmission of embryonic induction across a millipore filter6,8. In one of the best documented transfilter embryonic induction experiments, the inductive influence was appreciably restricted using filters 70 #m × 0.5/~m or 25 #m × 0.1 # m (ref. 3). The diffusible "induction molecules" under these conditions would require the rather implausible diameter of 0.1 #m or greater to explain these data. Mesenchymal cell processes infiltrating within the filter might provide an explanation; however, it has repeatedly been shown that direct contact between heterotypic tissues is not required for embryonic inductiona, 7. The presence of R N A within the intercellular organic matrix during epithelialmesenchymal interactions associated with tooth development 11,12, and the finding of several morphologically different matrix vesicles within this region, prompted our laboratory to initiate studies to test whether some of these vesicles contained RNA. Using the indium trichloride staining method for the ultrastructural localization of nucleic acids 4a, we observed that some vesicles demonstrated an appreciable affinity for the indium. This localization of indium within matrix vesicles was ribonuclease labile (unpublished observations). Preliminary experiments to isolate the intercellular matrix (Fig. 17) and subsequently to isolate the matrix vesicles from within this region, indicate that some matrix vesicles contain an " R N A - p r o t e i n complex ''44. In addition to these possibilities, other matrix vesicles may serve to transport Mech. Age. Dev., 1 (1972) 139-161

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organic or inorganic materials into the extracellular region for the formation of the dentine matrix. Increasing numbers of investigations have provided ultrastructural information obtained from other tissues which calcify to enhance such speculations14-18, 4~-47. Several studies describing chondrogenesis and osteogenesis indicated that the initiation sites for organic matrix mineralization are primarily associated with the appearance of matrix vesicles presumed to form from chondrocytes or osteoblasts15,18, 46. Possibly there are comparable functions for matrix vesicles during early dentinogenesis (Figs. 15 and 16). On the basis of our morphological evidence and the enlarging literature concerning matrix vesicles, several possible functions can be postulated: (1) the transport of structural molecules synthesized within each cell type for the formation of the extracellular organic matrices; (2) a mechanism by which enzymes can be transported within vesicles into the extracellular milieu for subsequent functions48; (3) a mechanism for the concentration of ions for subsequent calcification14; and (4) a mechanism for transferring developmental information between heterotypic ceils before cell division ceases in inner enamel epithelium and adjacent preodontoblasts. Each of these postulated vesicle functions requires additional experimentation. It is quite apparent, in our opinion, that the contents of these extracellular matrix vesicles must be chemically characterized. We are now attempting to isolate systematically these matrix vesicles and separate them on the basis of size and density in the hope of determining their compositions and functions. ACKNOWLEDGEMENTS This investigation was supported by U.S. Public Health Service Research G r a n t DE-02848-03 and Training Grant DE-00094-10 from the National Institute of Dental Research. Dr Slavkin is a recipient of a Research Career Development Award, 1-K4-DE-41739, and Dr Bavetta is a recipient of Research Career Award, 5-K06-DE-06083, U.S. Public Health Service, National Institute of Dental Research. The authors wish also to thank Mrs Joanne Leynnwood and Miss Susan Ibara for their technical assistance.

Fig. 17. A representative electron micrograph of the isolated, acellular intercellular matrix. Following dissection of 25-day embryonic New Zealand white rabbit incisor tooth primordia, dental papilla (pulp) was removed, the germinative matrix region (10-20/~m in thickness) was isolated, and this matrix was then sonicated for 15 seconds in a calcium- and magnesium-free, phosphate buffered solution at pH 7.4. Note the numerous matrix vesicles (arrows) retained following preparative procedures and the apparent removal of all cell processes. Compare this preparation with the descriptions in Figs. 4-6. For orientation, the inner enamel epithelium was located in the upper right region prior to microdissection and sonication. A mesenchymal cell process occupied the lower right region. × 47 000. Mech. Age. Dev., 1 (1972) 139-161

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