Cells and Materials Cells and Materials

Volume 3 Number 1 Article 6

1993

Mineralized Matrix Synthesis by Isolated Mouse Odontoblast-like Mineralized Matrix Synthesis by Isolated Mouse Odontoblast-like

Cells In Vitro Cells In Vitro

P. B. Andrews University of Toronto

A. R. Ten Cate University of Toronto

J. E. Davies University of Toronto

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Recommended Citation Recommended Citation Andrews, P. B.; Ten Cate, A. R.; and Davies, J. E. (1993) "Mineralized Matrix Synthesis by Isolated Mouse Odontoblast-like Cells In Vitro," Cells and Materials: Vol. 3 : No. 1 , Article 6. Available at: https://digitalcommons.usu.edu/cellsandmaterials/vol3/iss1/6

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Cells and Materials, Vol. 3, No. 1, 1993 (Pages 67-82) 1051-6794/93$5.00+ .00 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA

MINERALIZED MA TRIX SYNTHESIS BY

ISOLATED MOUSE ODONTOBLAST-LIKE CELLS IN VITRO

P.B. Andrews•, A.R. Ten Cate, J.E. Davies1

Faculty of Dentistry and 1Centre for Biomaterials, University of Toronto, 124 Edward Street, Toronto, Ontario, Canada, MSG 1G6

(Received for publication May 12, 1992, and in revised form March 31, 1993)

Abstract

First mandibular molar tooth germs were dissect­ed from 17 day mouse embryos. The dental papilla was isolated using both mechanical separation and enzymatic digestion. The cells of the papilla were then enzymatically disaggregated and cultured in 35 mm polystyrene dishes containing alpha minimum es­sential medium supplemented with 15 % fetal calf se­rum, 50 µglml ascorbic acid, 1 o-8 M dexamethasone and 10 mM Na-13-glycerophosphate. The cultures were maintained for 23 days. The cultured cells ini­tially appeared as large flat cells having numerous cell processes. Multilayered cell nodules, distributed randomly in the cultures, were apparent after 5 days. Matrix was visible within these nodules after 15 days . At day 23, histochemical staining identified alkaline phosphatase activity and von Kossa stained mineral­ized matrix localized to the cell nodules . Ultrastruc­turally, the cultured cells displayed classic character­istics associated with the odontoblast phenotype. Electron diffraction pattern analysis and energy dis­persive X-ray microanalysis, of the mineralized ma­trix, identified the co-localization of Ca and P in the form of hydroxyapatite crystallites. These results in­dicate that it is possible to grow mouse embryonic dental papilla cells, in vitro, and that these cells will form multilayered nodules of odontoblast-like cells which produce a mineralized matrix.

This reproducible method of cell culture lends it­self not only to the study of odontoblast differentia­tion, but also the development of in vitro methods of assessing the biological effects of both restorative materials and other chemical agents.

Key Words: Odontoblast differentiation, cell cul­ture, mouse, dexamethasone, ascorbic acid, 13-glyc­erophosphate.

•Address for correspondence: P .B. Andrews, Department of Paediatric Dentistry, Faculty of Dentistry, University of Toronto, 124 Edward St., Toronto, Ontario, Canada MSG 1G6 Phone no.: 416-979-4315 I FAX no. : 416-979-4566

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Introduction

Analysis of the processes involved in the differ­entiation of preodontoblasts is difficult either in vivo or in vitro, since the dental papilla consists of an het­erogeneous cell population. Thus, the establishment of a reliable cell culture system would facilitate study of not only the effects of various potential inductive biological agents, but also the effects of non-biologi­cal materials on differentiating odontoblasts. Indeed, odontoblasts form the focus of dental biocompatibility assays, since they are highly specialized cells which form a barrier between the dentin and the pulp and possess the capacity to synthesize reparative dentin. Most restorative procedures involve the exposure of dentin and odontoblast processes whose vitality may be compromised, or biological activity affected, by restorative materials. Thesleff (1986) described a method of enzymatic tissue separation, cellular disag­gregation and monolayer cell culture of preodonto­blasts. However, the cultured cells did not express the phenotypic characteristics associated with differ­entiated odontoblasts. Demonstration of phenotypic expression is essential in in vitro biocompatibility as­says, and potentially distinguishes them from meth­ods, including national and international standards to assess restorative dental materials (Browne, 1988), which rely on cytotoxicity assays.

We report the development of a reproducible cell culture protocol which permits preodontoblasts to dif­ferentiate, and secrete a collagen rich extracellular matrix which subsequently undergoes hydroxyapatite mineralization, in the absence of a basement mem­brane or epithelial tissues. Furthermore, we propose that this in vitro system provides the basis of a bio­compatibility assay for restorative dental materials.

Since the developmental processes involved in the differentiation of odontoblasts have not yet been fully elucidated, we initially review the extensive literature which indicates that epigenetic cell interactions are involved in an embryological cascade including induc­tion, competence, differentiation and determination.

P.B. Andrews, A.R. Ten Cate, J.E. Davies

Odontoblast Differentiation

The developmental processes involved in the dif­ferentiation of odontoblasts from the ectomesenchy­mal cell condensation of the dental papilla have not yet been fully elucidated. It is generally accepted that developmental cell interactions are involved in a cascade including determination , competence, induc­tion, and differentiation (Ruch, 1987). A major problem in developmental biology is to identify the controlling mechanisms involved in the expression of the mature phenotype of a given cell type (Ruch, 1987).

The temporal and spatial parameters characteriz­ing the development of the mouse first mandibular molar tooth germ have been extensively reported from the results of both in vivo and in vitro investiga­tions (Cohn, 1957; Hay, 1961; Glasstone, 1967; Kollar and Baird, 1969; Ahmad and Ruch, 1987). Initiation of odontogenesis occurs at day 11 of gestation and a minimum of 14-15 cell generations may exist prior to the cytodifferentiation of the first odontoblasts at day 18 (Zajicek and Bar-Leo, 1971; Ruch et al., 1973; Ruch and Karcher-Djuricic, 1975; Croissant et al., 1975). The expression of the odon­toblast phenotype is characterized by withdrawal from the cell cycle, elongation of the cell, eccentric posi­tioning of the nucleus, an accumulation of synthetic organelles in the cytoplasm with subsequent secretion of a collagen rich extracellular matrix that undergoes hydroxyapatite mineralization (Ruch et al., 1976, 1982; Lesot et al., 1982). At day 17, no differentiat­ed odontoblasts have developed and thus the dental papilla consists only of preodontoblasts (Cohn, 1957; Hay, 1961; Glasstone, 1967; Karcher-Djuricic et al., 1978; Yamada et al., 1980; Hurmerinta et al., 1980; Sakakura et al., 1984; Thesleff, 1986) . Therefore, the 17 day mouse first mandibular molar tooth germ represents an ideal stage in the developmental process for analyzing the mechanisms controlling the expres­sion of the odontoblast phenotype (Koch, 1967; Kollar and Baird, 1969; Slavkin, 1974; Thesleff and Hurmerinta, 1981; Ruch, 1984).

Several external factors are generally accepted as being necessary to bring about odontoblast differenti­ation. The first factor is the necessity for the neural crest cells to come into contact with ectoderm as they migrate to their first arch location (Lumsden, 1984). The second factor is the apparent need for the inter­nal dental epithelial cells of the dental organ to se­crete a basement membrane to initiate the final differ­entiation of the odontoblasts (Karcher-Djuricic, et al., 1978; Osman and Ruch, 1981).

68

Cell and Organ Culture Studies

In order to attempt to identify possible inductive agents of odontoblast cytodifferentiation, the compo­sition of the basement membrane has been invest igat­ed by cytochemistry, histochemistry, autoradio gra­phy, indirect immunofluorescence and biochemistry. The results of these studies indicate the presence of laminin, fibronectin, collagens type IV, type I, type I trimer, type III, heparin sulfate, chondroitin sulfate, and hyaluronate (Thesleff, 1976; Thesleff et al., 1981; Lau and Ruch, 1983). In order to examine the role of various substances as inductive agents in odontoblast differentiation, organ cultures of (enzy­matically isolated (trypsin) dental papillae have been used. However, attempts to induce odontoblast dif­ferentiation in vitro with gels containing collagen types I, II, III, and proteoglycans, individually or in combination, have not been successful (Koch, 1975; Thesleff et al., 1978). More recently, studies utiliz­ing extracellular matrix components deposited on Mil­lipore filters including collagen types I, II, III, V, laminin, fibronectin, hyaluronate, chondroitin 4 and 6 sulfates (isolated or mixed) failed to induce differ­entiation of preodontoblasts (Lesot et al. , 1984; Cam et al., 1987; Tziafas et al., 1988).

Methods and Materials

Collection of tooth buds

Laboratory raised Swiss mice were anesthetized at day 17 of gestation (vaginal plug equals day zero) using fluorothane in a bell jar followed by 0.1 cc ketamine 100 mg/ml intramuscularly, 0.1 cc pento­barbital 0.065 mg/ml intraperitoneal, 0.1 cc atropine 0.03 mg/ml subcutaneously. A total of six Swiss mice were dissected on three separate occasions pro­ducing 48 embryos and 96 first mandibular molar tooth germs. Approximately 5 % of the tooth germs were damaged during the dissection procedure and were therefore discarded. A more thorough account of these procedures has been previously published (Andrews, 1990).

Aseptic techniques were employed for all dissec­tion and culture procedures. Laparotomy was per­formed to expose and visualize the uterine chain of embryos. Starting at the distal end of the uterine horn, a single embryo was carefully dissected out of the uterine decidua, the trophoblast shell and Reichert's membrane were then removed. The em­bryo was staged according to the external develop­mental features, only Theiler day 17 embryos were used (Theiler, 1983). The mandible was removed at the level of the temporomandibular joint under a

Odontoblast culture system

stereoscopic dissecting microscope with a fibre optic light source.

The first molar tooth germs were then gently dis­sected free of the surrounding tissues and placed in 200 µl of alpha minimum essential medium (MEM) (Gibco Laboratories, Grand Island, N.Y., USA) on ice. These procedures were repeated with the re­maining embryos of the litter.

Trypsin separation

The dissected papillae were placed in 1 % trypsin (Type XII-S Sigma, St. Louis, Mo., USA) in Hank' balanced salt solution (HBSS) for 90 minutes at 4 ° C in order to digest the attachment of the basement membrane to the dental papilla while maintaining the attachment of the basement membrane to the dental organ (Osman and Ruch, 1981). The dental papilla and dental organ were transferred to 25 µl of MEM and mechanically separated using 30 g needles under an inverted phase contrast microscope.

Cell culture

The isolated dental papillae were transferred to 0.1 % trypsin (Type XIl-S, Sigma) in HBSS for 30 minutes at 37°C and then placed in culture medium consisting of MEM supplemented with 15 % fetal calf serum (Gibco). The tissues were shaken vigorously and passed through a sterile 20 µm nylon filter in order to produce a single cell suspension. The cells were plated on 35 mm tissue culture treated polysty­rene dishes at a density of approximately 250,000 cells per dish. The cell density was determined using an hemocytometer. The plated cells were cultured in 1 ml of culture medium consisting of MEM supple­mented with 15 % fetal calf serum, penicillin 1 mg/ml (Sigma), gentamycin 500 µglml (Flow Laboratories, McLean, Va., USA), amphotericin-B 3 µg/ml (Sig­ma), ascorbic acid 50 µglml (BDH Laboratories, Toronto, Ont., CAN), dexamethasone 10-8 M (Sig­ma), Na-Jj-glycerophosphate 10 mM (BDH) (Bellows et al., 1986). The cultures were maintained for 23 days at 37°C temperature, 5 % C02 atmosphere and 100 % humidity. After 12 hours, to allow settling and attachment to occur, the unattached cells were re­moved with the first medium change. Subsequently, the culture medium was changed every 48 hours. Monitoring was done daily using phase contrast microscopy .

Histological examination

Hematoxylin & eosin (H&E) staining: Intact tooth buds, and isolated dental papillae selected as controls, were fixed in Bouin's fixative, paraffin em­bedded, serially sectioned at 5 µm and stained with Harris's hematoxylin & eosin (Ambrogi, 1960). The

69

tissues were examined using transmission light microscopy.

Von Kossa staining: Some cultures, fixed with periodate-lysine-paraformaldehyde at day 23 of pri­mary culture, were washed in distilled water, and then incubated for 30 minutes with 5 % silver nitrate solution, rinsed in distilled water and incubated in 5 % sodium thiosulfate solution for 2-3 minutes, to localize phosphate and calcium salts (Cameron, 1930; Tenenbaum et al., 1992). The stained cultures were examined using an inverted phase contrast micro­scope, as a counterstain was not used.

Histochemical examination

Alkaline phosphatase staining: Cultures were fixed with periodate-lysine-paraformaldehyde (McLean and Nakane, 1974), for 15 minutes, at day 23 of primary culture. The cultures were then washed in distilled water, and incubated for 10 min­utes with sodium naphthyl phosphate in Tris buffer, pH 10, in the presence of Fast Red Violet (Drury and Wallington, 1967). The stained cultures were exam­ined using a Nikon Diaphot inverted phase contrast microscope in order to visualize the non-stained cells, as a counterstain was not used.

Transmission electron microscopy and electron diffraction

Cultures were fixed at day 23 of primary culture using 2.5 % glutaraldehyde in 0.2 M cacodylate buf­fer, pH 7. 3, at room temperature for 4 hours, washed in the same buffer and postfixed with 1 % osmium te­troxide in 0.2 M cacodylate buffer for 2 hours at room temperature. After washing in the same buffer, the cultures were dehydrated and stained using 3 % uranyl acetate in 70 % ethyl alcohol for 1 hour at room temperature, dehydrated and embedded in Epox 812 (Fullman Inc., Schenectady, N.Y., USA). Sec­tions, about 60-80 nm thick (silver-silver gold inter­ference colour), were cut using a Sorval 5000MT ultramicrotome, mounted on copper grids and exam­ined in a Philips 400T transmission electron micro­scope operated at 60 kV. Some ultrathin sections were examined for electron diffraction in a Philips EM430 transmission electron microscope operated at 100 kV. Energy dispersive X-ray microanalysis was performed using a Link AN 10, 000 system.

Results

Examination of controls confirmed the success of the dissection technique in isolating intact tooth germs (Figure la) and of the separation technique in dissociating the dental organ from the dental papilla (Figure lb).

P.B. Andrews, A.R. Ten Cate, J.E. Davies

Figure 1. (a) Control 17 day mouse first mandibu­lar molar tooth germ, outer dental epithelium (0), stellate reticulum (S), inner dental epithelium (I), basement membrane (B) , preodontoblasts (P), dental papilla (D). (b) Control 17 day mouse first mandi­bular molar dental papilla. Note the lack of dental organ or basement membrane with the outer surface of the dental papilla consisting only of preodonto­blasts. H&E stained sections. Field W,idths (F. W.): 1 mm.

Figure 2. Initial monolayer of cultured preodonto­blast cells 12 hours after initial plating after the first medium change showing the majority of remaining cells as large flat well spread cells with numerous thick cytoplasmic processes extending from a highly convoluted cytoplasmic skirt. Many of the processes end in a relatively optical dense area (arrowheads). Large nuclei containing prominent nucleoli are also apparent. Phase contrast photomicrograph. F. W.: 300 µm.

At 12 hours, the attached cells appeared as large well spread cells with numerous thick cytoplasmic processes extending from a highly convoluted cyto­plasmic skirt. Many of the processes ended in a relatively optical dense area (Figure 2). Some isolated cells were observed to produce a single predominant cell process with numerous lateral extensions and terminal branches . Some spindle shaped cells were also apparent at this stage, how­ever, they became less apparent with increasing time in culture.

70

@

After approximately 5 days in culture, some cells undergoing proliferation produced randomly located multilayered cell nodules. The central portion of the nodules appeared as a relatively optical dense region due to overlapping of the cytoplasmic skirts of adja­cent cells. The cell nuclei, therefore, became the most prominent feature of these cells. At the periph­ery of the nodules, where the cytoplasmic skirts were no longer overlapping, the characteristic cytoplasmic processes with numerous lateral and terminal branch­es were apparent.

The number of cells increased with time in cul­ture producing large areas of confluent cells contain­ing multilayered regions . Using phase contrast light microscopy, matrix production was evident within the

Odontoblast culture system

Figure 3. Phase contrast photomicrographs showing alkaline phosphatase staining and the von Kossa reaction product localized to the multilayered regions within large areas of confluent cells. (a) Alkaline phosphatase staining clearly localized to the cells in the multilayered region. (b) Alkaline phosphatase staining specifically associated with the cells of the nodule indicating the site specific nature of the mineralization process. (c) Von Kossa reaction product localized to the extracellular matrix associated with two different nodules. Note the dif­ference in degree of maturation based upon amount of matrix produced. Also note the fenestrated appearance of the matrix. (d) The mineralized matrix appears to be deposited around the periphery of individual cells pro­ducing a highly fenestrated appearance. F.W.: (a and c) 165 µm; (band d) 65 µm.

multilayered regions by 15 days in culture. This matrix appeared optically dense, contained numerous fenestrations and appeared to be deposited around the periphery of the individual cells.

At day 23, the randomly located multilayered cell nodules contained cells staining positively for alkaline phosphatase activity and the von Kossa reaction prod­uct could be localized to the highly fenestrated

71

extracellular matrix (Figure 3).

Cell ultrastructure

Ultrastructurally, the characteristics displayed by the cells, making up the nodules, were observed to be similar in all sections examined. The cells had a well characterized shape with a columnar basal cell body 1-2 µm wide and an elongate tapering cell process (Figure 4a) .

P.B. Andrews, A.R. Ten Cate, J.E. Davies

The total length of the cells was approximately 20 µm. The distal end of the process was very nar­row measuring approximately 0.1 µmin width. The nucleus, oval in shape and smooth in contour, was eccentrically positioned at the widened basal end of the cell (Figure 4b). The cytoplasm proximal and

72

lateral to the nucleus appeared relatively electron lucent due to a paucity of cytoplasmic organelles. A large Golgi area was densely packed with numerous Golgi elements and cytoplasmic bodies. Immediately distal to the nucleus and surrounding the Golgi area, an abundance of cistemae of agranular and granular

Odontoblast culture system

Figure 5. (a) Nascent sites of mineralization are typically observed in association with collagen fibrils that are oriented parallel with the long axis of the odontoblast-like cells along the base of the dish. Additional sites of mineralization are observed in the extracellular compartment between the cells associated with collagen fibrils. The mineral appears to advance along the length of the collagen fibrils (arrow). (b) As the mineralization ad­vances an irregular surface is caused by the varying lengths of the collagen fibrils. An initial non-collagenous poorly mineralized 0.1 µm thick layer is always secreted along the base of the dish (D). (Compare Figure 4, Sa, Sb and 6). F.W.: (a) 1.4 µm; (b) 2.1 µm.

Figure 4. (a) Montage of an odontoblast-like cell after 23 days in primary culture. Note that the distribution of the mineralized matrix is, for the most part, random although some directionality is evident (top left). At higher magnifications of similar areas (Figures 5 and 6), this directionality of mineralization is associated with the presence of cross-banded collagen fibres. (b) Magnified view of the columnar basal end of the odontoblast­like cell with eccentric nucleus. (c) Magnified view of the centrally located, well developed, synthetic cytoplas­mic organelles. The nucleus (N) is positioned eccentrically in the columnar basal area of the cell. The organelles are fairly well developed in the cytoplasm distal to the nucleus. The Golgi area (G) is close to the nuclear region surrounded by large amounts of granular endoplasmic reticulum (E) and vesicles (V). The ends of the cell con­tain few organelles. Numerous minute cytoplasmic extensions projecting into the extracellular compartment are evident (arrowheads). A continuous band of mineralized matrix (M) is present along the base of the culture dish (D) with an initial non-collagenous poorly mineralized layer. Nascent areas of mineralization are also present between cells associated with the collagen containing extracellular matrix (C). Figure height in (a) 25 µm; figure widths (b and c) 6.2 µm.

endoplasmic reticula were observed. The endoplas­mic reticulum formed long strands of cisternae paral­lel to each other and to the long axis of the cell. There was an abundance of mitochondria and vesicles with a granular content in this area (Figure 4c). Some vesicles were fused with the plasma membrane. The tapering cell process was relatively electron

73

lucent being composed of a fine granular cytoplasm with few organelles or cytoplasmic bodies. Numer­ous minute cytoplasmic projections were observed extending into the extracellular compartment. The cell membranes of adjacent cells were attached to each other at different levels by gap junctions.

P.B. Andrews, A.R. Ten Cate, J.E. Davies

Figure 6. Transmission electron photomicrograph to show the interface formed between the calcified extracellular matrix produced by the odontoblast­like cells, and the polystyrene tissue culture dish (D). There is an initial afibrillar layer, exhibiting an undulating superior aspect, which is clearly demarcated from the collagen containing calcified matrix above. The latter is formed by the fusion of spherical foci of mineralization, associated with collagen fibrils, as seen in the extracellular com­partment immediately inferior to the overlying cell. F.W.: 1 µm.

Figure 7 (below). (a) Energy dispersive X-ray microanalysis displaying characteristic peaks for calcium (Ca) and phosphorus (P). Note the co­localization of the Ca/P in the mineralized matrix associated with the odontoblast-like cells. (b) Electron diffraction pattern of the extracellular matrix associated with the odontoblast-like cells, which exhibits reflections (arrowheads), with de­creasing relative intensities, corresponding to 2. 74 (outer most ring), 3.03 and 3.44 A d-spacings characteristic of hydroxyapatite crystallites.

X-RAY ('~_-Live: 100s Preset: 100s Remaining: , , Rea. l: 127s 21 % Dead

Ca.

p

< • 0 5. 1 20 I< e:V FS= 2K ch 266= MEM1:EOS1 OOONTOBLAST MATRIX

Extracellular matrix

10.2 > O ct,s

The extracellular compartment comprised a fine granular amorphous ground substance with an abun­dance of collagen fibrils oriented roughly parallel to the long axis of the cell (Figure 5 and 6). Areas of mineralization were typically found associated with large collagen fibrils. Mineralization appeared to be progressing along the length of these fibrils. A con-

74

®

sistent finding was the presence of a band of mineral­ized matrix along the surface of the culture dish, which, after 23 days of primary culture, was approxi­mately 0.3-0.5 µm thick (Figures 4-6). Furthermore, there was an electron lucent area that consistently appeared as the initial 0.1 µm of matrix was pro­duced (Figures 4-6). Although it appeared that the cells making up the nodules were separated from the culture dish, it is possible that contact may have

Odontoblast culture system

occurred via cytoplasmic projections through the min­eralized matrix.

Energy dispersive X-ray microanalysis produced characteristic peaks for P and Ca obtained by com­puter subtraction of image sets (Figure 7a). Electron diffraction pattern analysis identified reflections, with decreasing relative intensity, corresponding to JCPDS (Joint Committee on Powder Diffraction Standards) 2.74, 3.03 and 3.44 A d-spacings characteristic of hydroxyapatite crystallites (Figure 7b).

Discussion

The developing tooth germ consists of an hetero­geneous population of cell types and extracellular en­vironments with potentially confounding influences. In order to study the parameters that affect odonto­blast differentiation, matrix production and mineral­ization, it would be advantageous to develop a repro­ducible preodontoblast cell culture protocol that can be stimulated to produce odontoblast-like cells and an hydroxyapatite mineralized extracellular matrix. Sup­port for the odontoblastic nature of our cultures is de­rived from the isolation procedure, morphologic char­acteristics, and functional characteristics of the cul­tured cells. Day 17 mouse first mandibular molar tooth germs were utilized. At that stage of develop­ment the dental papilla consists only of preodonto­blasts (Cohn, 1957; Hay, 1961; Glasstone, 1967; Karcher-Djuricic et al., 1978; Yamada et al., 1980; Hurmerinta et al., 1980; Sakakura et al., 1984; Thesleff, 1986). Uncontaminated isolation of the dental papilla is an essential prerequisite in harvesting preodontoblasts. The successful use of trypsin to di­gest the attachment of the basement membrane to the dental papilla while maintaining the attachment of the basement membrane to the dental organ has been ex­tensively reported (Koch, 1967; Kollar and Baird, 1969; Ruch et al., 1976; Karcher-Djuricic et al., 1978; Osman and Ruch, 1981; Olive and Ruch, 19 82). Furthermore, the use of trypsin digestion fol­lowed by mechanical separation of the dental papilla from the dental organ does not alter mitotic activity of preodontoblasts at the periphery of the dental pa­prnla (Olive and Ruch, 1982), nor the terminal differ­enttiation of these cells to become odontoblasts (Kollar and Baird, 1969; Olive and Ruch, 1982). Exami­naltion of controls confirms the success of the enzy­matic and mechanical separation techniques used in ou.r system to isolate the dental papilla from the epi thelial tissues and basement membrane.

Isolated cells

The release of the preodontoblasts from the den-

75

tal papilla was again achieved by enzymatic (trypsin) and mechanical disaggregation to produce a single cell suspension. Thesleff (1986) using a similar pro­cedure compared the cultured preodontoblasts to gin­gival mesenchyme and undifferentiated mandibular mesenchyme. The cultured preodontoblasts were de­scribed as large flat cells with numerous cellular extensions while the other mesenchymal cell popula­tions displayed a spindle shape typical of fibroblasts. However, the preodontoblasts persisted as a mono lay­er and failed to express the phenotypic characteristics of differentiated odontoblasts (Thesleff, 1986). In our cultures, the preodontoblasts initially formed a monolayer and displayed a similar morphology to those described by Thesleff exhibiting large well spread cells with numerous thick cytoplasmic process­es extending from a highly convoluted cytoplasmic skirt. Thus, the isolation procedure and the culture conditions utilized in this protocol clearly favour the establishment of preodontoblast cells in primary cul­ture. Furthermore, the matrix producing cells are representative of these differentiating cells in tooth germs rather than reprogrammed dental mesenchymal cells which are associated with the secretion of reparative dentin.

The presence of multilayered nodules indicates the importance of cell density in cytodifferentiation, matrix production and mineralization in vitro. The importance of multilayered nodules has previously been reported in mature pulp cell cultures (Naka­shima, 1991) and numerous bone cell culture systems (Sudo et al., 1983; Whitson et al. , 1984; Nefussi et al., 1985; Bellows et al., 1986; Ecarot-Charrier et al., 1988). Odontoblast-like cells were observed only inside the nodular multilayered parts of the cultures. Genetically determined cells, making up the nodule that had undergone an appropriate number of cell cycles, would be competent, given the appropriate extracellular environment, to undergo cytodifferentia­tion, while the daughter cells would continue to undergo proliferation.

The expression of the odontoblast phenotype can be described based on both morphologic and function­al characteristics and include withdrawal of the pre­odontoblast from the cell cycle, elongation, polariza­tion, and ultimately extracellular mineralized matrix production (Ruch et al., 1976). Polarization is de­pendent on certain cytoskeletal changes. The cell be­comes larger, the nucleus takes up an eccentric posi­tion, and the apical part of the cell forms the odonto­blast cell process (Ruch et al., 1982). The ultra­structural characteristics associated with the odonto­blast phenotype have been reported extensively (Garant et al ., 1968; Takuma and Nagai, 1971;

P.B. Andrews, A.R. Ten Cate, J.E. Davies

Katchburian, 1973; Baume, 1980; Heritier et al., 1989; Arsenault and Robinson, 1989) and include the accumulation of synthetic organelles and the develop­ment of gap junctions.

After 23 days in primary culture the cells making up the multilayered nodules exhibited a number of ul­trastructural features associated with the odontoblast phenotype, such as a columnar cell body, an elongate tapering cell process, an eccentrically positioned nu­cleus, accumulation of synthetic organelles in the cytoplasm, and gap junctions between the cells.

Odontoblast differentiation and mineralized matrix production

Functional differentiation of odontoblasts implies alkaline phosphatase activity, matrix vesicle produc­tion, and the secretion of a collagen rich extracellular matrix and its mineralization. The extracellular com­partment in our cultures comprised a fine granular amorphous ground substance with an abundance of banded collagen fibrils oriented roughly parallel to the long axis of the cell, which is similar to the rela­tionship seen in coronal mantle dentin. However, in vivo, the site and geometry of the matrix elaborated by differentiating odontoblasts is influenced by the presence of the basement membrane (Magloire et al., 1992; Thesleff and Vaahtokari, 1992). Thus, the api­cal secretion, and attendant directionality of the elab­orated matrix, typical in in vivo development, would not be expected in our culture system.

In our cultures, alkaline phosphatase activity and von Kossa reaction product were localized only to the multilayered cell nodules supporting the concept of site specific extracellular mineralized matrix produc­tion. Similar results have been described in the bone literature using the same culture conditions (Ecarot­Charrier et al., 1983; Sudo et al., 1983; Whitson et al., 1984; Nefussi et al., 1985; Bellows et al., 1986; Maniatopoulos et al., 1988). Matrix vesicles were not observed in the sections examined from our cul­tures. In vivo, matrix vesicles are apparent during the initial stages of mineralization referred to as the pre-calcification and incipient calcification stages of development (Katchburian, 1973). Mineral deposi­tion during the advanced stage of calcification most likely proceeds by a nucleation phenomenon along the long axis of collagen fibres (Katchburian, 1973; Christoffersen and Landis, 1991). Our cultures were examined ultrastructuraily after 23 days in primary culture. At that stage, large tracts of mineralized matrix were observed not only along the base of the polystyrene culture dish, but also in association with large bundles of banded collagen in the extracellular space of the multilayered nodules. In vivo, the latter

76

would correspond to the advanced stage of calcifica­tion, but not the pre-calcification or incipient calcifi­cation stages of development. This may explain the lack of matrix vesicles and the abundance of collagen associated mineralization observed in our cultures, al­though it is possible that these could have occurred at earlier stages in the culture.

The temporal and spatial ultrastructural character­istics associated with the initial mineralization of dentin in vivo, have previously been reported (Arsenault and Robinson, 1989; Christoffersen and Landis, 1991). Nascent sites of mineralization are typically found in association with collagen fibrils. With further development, larger tracts of mineral­ized dentin are elaborated. As the dentin mineral ad­vances, an irregular surface is caused by the varying lengths of the projecting terminal ends of the mineral­ized collagen fibrils (Arsenault and Robinson, 1989). In our cultures, after 23 days of primary culture, similar nascent sites of mineralization could be seen in the amorphous matrix association with large colla­gen fibrils. With further development, larger tracts of mineralized dentin are elaborated. As the dentin mineral advances, an irregular surface is caused by the varying lengths of the projecting terminal ends of the mineralized collagen fibrils (Arsenault and Robinson, 1989). In our cultures, after 23 days of primary culture, similar nascent sites of mineraliza­tion could be seen in the amorphous matrix associated with large collagen fibrils. However, it is clear that mineralization occurred not only within the collagen rich extracellular matrix, but also on the surface of the polystyrene tissue culture dish. This surface de­position of mineral was seen only beneath forming multilayered nodules and not ubiquitously in the cul­ture dish. It is noteworthy that a similar cement-like substance has recently been reported by Beertsen and van den Bos ( 1990) who reported the production, in culture, of a mineral phase of unknown origin, com­prising "fine needle-shaped crystallites embedded in a granular matrix of moderate electron density" into which collagen fibrils were embedded. A similar cell mediated calcified matrix has recently been reported beneath forming bone nodules in vitro (Davies et al., 1991a). This matrix which preceded overt collagen fibre production, comprised an initial fine organic network into which calcium phosphate crystallites were seeded (Davies et al., 1991b). These thin layers (approximately 0.5 µm) correspond to the known ability of hard tissue forming cells to elabo­rate collagen-free mineralized matrices. Examples of such mineralized matrices in dental tissues include the afibrillar cement that has been observed in layers, about 100 nm thick, over the enamel at the cemento-

Odontoblast culture system

enamel junction (Listgarten, 1966) and intratubular dentin, the hypermineralized dentin which is slowly laid down within the dentinal tubules and ranges in thickness from 44-750 nm (Ten Cate, 1989). Simi­larly for bone tissue, initial elaboration of an appar­ently collagen free mineralized matrix is exemplified by the cement line which forms at the periphery of secondary osteons (Davies et al., 1991b).

Influences on odontoblast induction

An inductive influence for the basement mem­brane in odontoblast differentiation has recently been suggested (Koch, 1967, 1975; Kollar and Baird, 1969; Slavkin, 1974, 1982; Thesleff et al., 1978, 1981; Lesot et al., 1984; Tziafas et al., 1988). It is also probable that circulatory or other diffusible fac­tors operating over long distances are also involved in the regulation of odontoblast differentiation (Thesleff and Partanen, 1984). The processes involv­ed in odontoblast differentiation in vivo are likely mediated by nutritional, hormonal, and/or enzymatic factors, all acting in concert to create a specific mi­croenvironment. Unfortunately, attempts to stimulate odontoblast cytodifferentiation using basement mem­brane components and specific growth factors in vitro have been unsuccessful (Thesleff, 1976; Thesleff et al., 1981; Lau and Ruch, 1983; Thesleff and Partanen, 1984). Our results demonstrate that it is possible to culture isolated preodontoblast cells from mouse first mandibular molar tooth germs in the ab­sence of epithelial tissue or basement membrane and stimulate the preodontoblasts to undergo cytodifferen­tiation to become odontoblast-like cells and produce a collagen rich hydroxyapatite mineralized extracellu­lar matrix. Therefore, the popular concept that the terminal differentiation of the odontoblast requires the presence of the mesenchymal face of a time and space specific basement membrane (Karcher-Djuricic et al., 1978; Thesleff et al., 1978; Osman and Ruch, 1981) must be re-evaluated.

The expression of the odontoblast phenotype in our system may be related to cell density and cell-cell interactions and/or the accumulation of important components in the extracellular matrix. A specific micro-environment may be created based on the in­clusion of ascorbic acid, dexamethasone and {3-glyc­erophosphate in the culture medium which permits competent cells tt> express the characteristics associat­ed with the odontoblast phenotype. Ascorbic acid was included based upon its role in the hydroxylation of collagen. The hydroxylation of certain prolyl and lysyl residues facilitates the formation of the triple helical structure of collagen and thereby encourages the synthesis of collagen rich extracellular matrix .

77

Ascorbic acid has also been found to encourage cell proliferation and to be essential for the maintenance of odontoblast differentiation (Barnes, 1975; Levenson, 1976; Schiltz et al., 1977). Dexametha­sone is a glucocorticoid which, when added to osteo­blast cultures on a short term basis, encourages dif­ferentiation (Canalis, 1983; Hahn et al., 1984; Tenenbaum and Heersche, 1985) and increases alka­line phosphatase activity of odontoblasts in culture (Karcher-Djuricic and Ruch, 1970). Green et al. (1990) have demonstrated that dexamethasone induces a rise in alkaline phosphatase gene transcription. The exact role of alkaline phosphatase in the mineraliza­tion process is not fully understood, although it is considered to be of primary importance. Alkaline phosphatase releases phosphate ions through hydroly­sis of phosphate esters (Ecarot-Charrier et al., 1988), it has also been proposed to destroy local inhibitors of mineralization, such as pyrophosphate (Fleish and Neuman, 1961), to act as a phosphate (Hsu and Anderson, 1977; Warner et al. , 1983) or a calcium­binding protein (de Bernard et al., 1985).

The rationale for the use of {3-glycerophosphate is that mineralization requires an adequate supply of phosphate ions. The initiation of mineralization may require an increase in the local concentration of in­organic phosphate. Organic phosphate may be the source of the necessary inorganic phosphate through enzymatic hydrolysis of phosphate esters by alkaline phosphatase (Tenenbaum and Heersche, 1982; Ecarot­Charrier et al., 1988). Therefore, a deficiency of organic phosphate may be the limiting factor in elicit­ing in vitro mineralization. In our culture system, {3-glycerophosphate may act as an organic source of phosphate ions. The phosphate ions would only be released in areas where alkaline phosphatase enzyme activity is present, thus allowing the initiation of mineralization in a physiologic site specific manner.

Concluding Remarks

As previously stated the expression of the odon­toblast phenotype is characterized by the following steps: withdrawal from the cell cycle, elongation of the cell, eccentric positioning of the nucleus, and ac­cumulation of synthetic organelles in the cytoplasm with subsequent secretion of a collagen rich extracel­lular matrix that undergoes hydroxyapatite minerali­zation (Ruch et al., 1976; Lesot et al., 1982; Ruch et al., 1982). Definitive identification of the odonto­blast phenotype is only possible through the identifi­cation of dentin specific proteins (Takagi and Veis, 1984; Butler, 1984; MacDougall et al . , 1985). Until antibodies to these proteins are more readily avail-

P.B. Andrews, A.R. Ten Cate, J.E. Davies

able, a definitive statement as to the odontoblastic nature of our cultured cells is not possible. Never­theless, we believe that the experimental evidence re­ported herein provides a clear indication of differen­tiation and extracellular matrix production by odonto­blasts in vitro. This culture system is, therefore, potentially suitable, not only for the investigation of induction and differentiation mechanisms in odonto­blasts, but also as a means of assessing the biocompa­tibility of candidate dental materials.

Acknowledgements

The authors are grateful to Doug Wagner for his expert technical assistance. The work reported is ex­tracted from an M.Sc. thesis of one of us (PBA).

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Odontoblast culture system

Discussion with Reviewers

J. Y. Ruch: Why were antibodies against dentin phos­phoprotein(s) not used? Are phosphoproteins specific markers of odontoblasts? A.L. Boskey: It has frequently been suggested that "o<lontoblast cultures" are more osteoblastic than odontoblastic. In the absence of demonstration of ex­pression of odontoblast specific proteins (e.g., phos­phophoryn, by either in situ hybridization or immuno­fluorescence}, do you think that this is true of your cultures? Authors: No, we do not think it is true that the cul­tures we describe are more osteoblastic than odonto­blastic. We base this argument on the work of Palmer and Lumsden [Palmer RM, LuJilsden AGS (1987) De­velopment of periodontal ligament and alveolar bone in homografted recombinations of enamel organs and papillary, pulpal and follicular mesenchyme in the mouse. Arch. Oral Biol. 32, 281-289], who showed that the originally pluripotential mesenchymal cells of the dental papilla undergo progressive determination, become restricted in their options, and lose their ca­pacity to differentiate into osteoblasts at the bell stage of tooth development. It is from developing teeth of this stage that we have extracted our cells. We men­tion, in our concluding remarks, that definitive identi­fication of the odontoblast phenotype is only possible through identification of dentin specific proteins. The dentin phosphoproteins would be obvious targets for such experiments. Indeed, we attempted to demon­strate dentin phosphoproteins using an antibody kindly donated by Professor Veis. However, at the time at which the work was carried out (Andrews, 1990) we failed to demonstrate labelling with the supplied anti­body to either our experimental, or positive control (rat incisor was recommended) specimens. Clearly, therefore, we have not reported these negative results especially as we know that, in the intervening period, the donor laboratory has achieved more reliable results with this antibody (A. Veis, personal communication). We would agree that it would now seem timely to re­peat these experiments in order to add biochemical data to the morphological evidence presented here.

Reviewer VI: I am puzzled by the reported lack of matrix vesicles in the cultures despite the presence of areas of unmineralized and early calcified matrix. Did the authors have the chance to look at long-term cul­tures grown without 13-glycerophosphate, which pre­sumably will not mineralize? A.L. Boskey: Do you think extracellular matrix vesi­cles were present in the matrix but are masked by the mineral? Do you see vesicle-like structures if you de­calcify the sections? Since matrix vesicles are seen in mantle dentin but not in more mature dentins are their

81

absence surprising? I. Thesleff: Could it be that in vivo, the vesicles originate from the epithelium or that the mineralization that is observed in vitro may not represent a physio­logical mineralization process? Authors: We do not report herein the lack of matrix vesicles as a phenomenon, but simply an experimental observation. Thus, the presence of matrix vesicles should not be considered an essential pre-requisite of mineralization. In some TEM sections we saw small, circular, membrane bound structures, although these were devoid of mineral at the resolution limits of our microscopes. We considered these to be cell filopo­dial extensions which had been cross-sectioned, but definitive exclusion of these structures as matrix vesi­cles would only be achieved by production, and three­dimensional reconstruction, of TEM serial sections, which we have not undertaken. Thus, we do not state that they are absent in these cultures, but rather, that we did not observe them. Similarly, one could guess that matrix vesicles may have been masked by the mineral. We did not produce demineralized sections from these preparations. However, matrix vesicles are normally identified at a mineralizing front; yet, in all the sections we examined we found no evidence of them at these locations. As matrix vesicles are char­acteristic of connective tissue matrices, for example cartilage, where there is no implication of involvement of epithelial components, we do not entertain the no­tion that epithelium may be implicated, in this manner, in dental tissues. We have not looked at long-term cultures grown without 13-glycerophosphate. From cultures of rat bone marrow cells which we use exten­sively to produce populations of differentiating osteo­genic cells (Davies et al., 1991a, b}, we find that min­eralization will occur in the absence of 13-glycerophos­phate, although the yield of mineralized matrix is low. In view of the number of examples of such matrices in vivo, and the site-specific nature of this formation in vitro, we believe that this does represent a physiologi­cal phenomenon.

A.L. Boskey: Why do you think mineral deposition appears on the polystyrene surface? Could it be due to the anaerobic environment at the bottom of the cul­ture plate? To altered pH associated with this envi­ronment? Could it represent random deposition due to the altered phosphate level (10 mM 13-glycerophos­phate, yields 8-10 mM Pi)? Authors: We should emphasize, as discussed in the text, that the mineralization at the culture dish surface was not ubiquitous; it only occurred beneath develop­ing multilayered cell nodules. Outside the nodular areas no mineralization was seen on the culture dish surfa~e. We are quite sure, therefore, that this calcifi-

P.B. Andrews, A.R. Ten Cate, J.E. Davies

cation does not represent a random deposition. In similar culture systems, we have monitored the devel­opment of mineralized nodules when gas-permeable polymer membranes or porous alumina ceramic plates are used as culture substrates. These substrates are designed to maintain the cells at the culture surface in an aerobic environment. Thus, we do not believe that the mineral deposition is due to an anaerobic environ­ment. The appearance of the mineral at the interface is discussed in the text although we would point out that there are several examples, in hard tissue biology, of similar thin mineralized matrices which are appar­ently free of assembled collagen fibres (see also, Davies et al., 199la, b).

J. V. Rueb: The effects of trypsin during dental-tis­sues separation is not clear: normally the basement membrane should be hydrolyzed. Authors: We have simply adopted what we consider to be a standard technique for this separation which has been widely reported and defended by others (Koch, 1967; Kollar and Baird, 1969; Ruch et al., 1976; Karcher-Djuricic et al., 1978; Osman and Ruch, 1981; Olive and Ruch, 1982).

I. Thesleff: I agree with the authors that generation of adequate cell density is probably critical. Might it be at least equally critical that, during the prolonged culture, the cells elaborate enough of a special type of extracellular matrix (plus growth factors?) which is necessary for the polarization of the odontoblasts and for the expression of the differentiated state? Authors: Yes. We feel that this is highly likely al­though the levels of factors produced would them­selves be related to cell density.

I. Thesleff: Could it be that the apparent controver­sy between the findings here (that odontoblasts seem to differentiate without epithelium) and the earlier pre­sented proposal that the epithelial basement membrane is crucial for differentiation, can, in fact, be ex­plained? And that the earlier hypothesis does not real­ly need real re-evaluation. It is quite apparent also from other earlier literature that odontoblasts may differentiate from the dental pulp cells later without obvious epithelial contact. Therefore, it can be speculated that during in.vivo development, the base­ment membrane is necessary as a mechanical support upon which the preodontoblasts align (this would be replaced by the culture dish) and on which the cells secrete special extracellular matrix molecules and growth factors. [The latter] may function as autocrine regulatory factors for the differentiating cells (see discussion in Thesleff and Vaahtokari, 1992). Authors: We agree entirely with this important point. However, the culture dish, in the manner employed,

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could not be expected to confer the directionality of either cell or matrix as is seen with the basement membrane in vivo.

H. Lesot: What is the population of preodontoblasts in the cultured dental papillae cells? Are all dental mesenchymal cells considered as potential preodonto­blasts? Authors: Depending upon the environment in which they find themselves, we consider all pluripotential dental mesenchymal cells to be potential preodonto­blasts. However, we are unable to speculate on the percentage of such cells in our culture system.

H. Lesot: Could it be shown that odontoblast-li.ke cells within the nodules are postmitotic or what is the population of cells still able to divide? Authors: No. Those cells involved in overt extracel­lular matrix production are assumed to be post-mitotic. However, we are unable to speculate on the percent­age of extra-nodular cells, in our cultures, which would still be capable of mitosis.

Reviewer VI: Do the cells need the (continuous) presence of dexamethasone in order to express the odontoblast phenotype in culture? If so, are the for­mation of multilayered nodules, the appearance of odontoblast-like cells and/or the production of a cal­cified matrix impaired in dexamethasone-free cultures? Authors: We have not carried out such experiments and, therefore, cannot provide a direct answer to this interesting question. However, from our discussion of the effects of dexamethasone and, in particular, as­suming the importance of alkaline phosphatase expres­sion, we feel the reference to the work of Green et al. (1991) is of particular significance. As each mineral­izing nodule represents a "time capsule" of differenti­ation events, in an analogous fashion to the bell stage of tooth development, there should always be cells, particularly at the periphery of the nodules, which need the presence of dexamethasone to facilitate their expression of the odontoblast phenotype. Thus, we would expect the withdrawal of dexamethasone during culture to diminish the yield of mineralizing nodules in this system, but not impair the mineralization which, at any one time point, has already begun.

Reviewer VI: Will the authors comment on the dif­ference in width between the non-mineralized and min­eralized collagen fibrils seen in Figure Sa? Authors: The collagen fibres (fibrils) are only visible in this photomicrograph when non-mineralized since the mineral completely masks the structure of, and therefore, the possibility of, identifying the fibres. With increasing time we would expect the non-miner­alized collagen to become masked by the elaboration of new mineral.