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In vitro mineralization of a three-dimensional collagen matrix by human dental pulp cells in the presence of chondroitin sulphate
Archs oral Biol. Vol. 35, No. 4, pp. 301-309,1990
0003-9969/90 $3.00+0.00 Press plc
Printed in Great Britain. All rights reserved
Copyright c 1990Pergamon
IN VITRO MINERALIZATION OF A THREE-,DIMENSIONAL COLLAGEN MATRIX BY HUMAN DENTAL PULP CELLS IN THE PRESENCE OF CHONDROITIN SULPHATE M. BOUVIER,‘,~*A. JOFFRE’ and H. MAGLOIRE’ ‘Laboratoire d’Histophysiologie et de Pathologie des Tissus Dentaires, Rue Guillaume Paradin, 69372, Lyon Cedex 8 and ZBIOETICA S.A., 32 rue Saint Jean De Dieu, 69007, Lyon, France (Received 3 May 1989; accepted 29 September
1989)
Summary-These matrices were used as cell culture substrates to investigate the influence of extracellular molecules on mineralization. Pulp cells seeded in type I collagen or type I collagen-chondroitin-4-sulphate sponges were able to grow and were morphologically similar to cells responsible for reparative dentine formation in tko. In sponges consisting of collagen only, the cells elaborated an abundant new matrix which became organized with time and consisted of collagen fibres surrounded by fibrillar material, but no mineralization was observed. In collagen-chondroitin sulphate sponges, cells deposited less and poorly organized mal.rix; in these, calcification occurred, increasing with time, and at the ultrastructural level, small needle-like crystals containing calcium and phosphorus were scattered throughout the sponge fibres. These observations suggest that chondroitin sulphate might influence in virro calcification induced by pulp cells. Key words: collagen, mineralization, chondroitin sulphate, dental pulp.
INTRODUCTION Dental pulp is a loose mesenchymal tissue, characterized by its particular location, and almost entirely enclosed in a mineralized tissue, dentine. Dentine matrix contains type I collagen associated with noncollagenous proteins (including phosphoproteins and Gla-proteins) and proteoglycans in which the predominant glycosammoglycan in numerous species is chondroitin sulphate (Linde, 1985). Dentine is elaborated by highly differentiated cells, the odontoblasts, located at the periphery of the pulp. Their terminal differentiation occurs in a specific temporo-spatial pattern mediated by epithelial-mesenchymal interactions (Thesleff aml Hurmerinta, 1981; Ruth et al., 1982). When a tooth become carious, a new mineralized scar tissue, reparative dentine, is elaborated by underlying differentiated pulp cells (Feit, Metelova and Sindelka, 1970; Fitzgerald, 1979; Magloire, Joffre and Hartmann, 1988). The origin of this induction is still unclear; epithelial-mesenchymal interactions appear not to be involved as epithelial cells and specific basement membrane components have not been detected. One approach to the study of reparative dentine formation consists of growing pulp cells in tissue culture (Zusman and Ioachim, 1964; Magloire and Dumont, 1976; Got’oh, Saito and Sato, 1979; Shuttleworth, Berry and Wilson, 1980; Magloire et nl., 1981). Partial cytodifferentiation of explanted pulp *Address correspondence to: M. Bouvier, Laboratoire d’Histologie, Facultb d’Odontologie, Rue G. Paradin, 69372, Lyon, Cedex 8, France.
cells has been demonstrated through modulation of protein synthesis (Magloire ef al., 1984), but no mineralization was observed. Further differentiation has been obtained by using components of the extracellular matrix as substrates for the cultured cells (Kleinman et al., 1987). Three-dimensional culture matrices consisting of native type I collagen supplemented with glycosaminoglycans or fibronectin have been shown to control the regulation of tendon and dermal fibroblast metabolism (Doillon and Silver, 1986; Doillon, Silver and Berg, 1987). We have now used threedimensional culture substrates consisting of components analogous to the dentine matrix to study whether human pulpal cells grown on these type I collagen or type I collagen-chondroitin-4-sulphate sponges could induce calcification. MATERIALS AND
METHODS
Collagen sponges were manufactured under patent by BIOETICA (Lyon, France) from the skin of young calves. In brief, the dermis had been separated, ground, extensively washed in phosphate buffer (pH 7.8) and in distilled water, swollen in dilute acetic acid, freeze-dried and reticulated by heat. The samples were sterilized by ethylene oxide. Collagenchondroitin-4-sulphate sponges were prepared as above but with the addition of chondroitin-4-sulphate before freeze-drying. Chondroitin sulphate was extracted from bovine nasal cartilage by alkaline treatment and ethanol precipitation, essentially as described by Taniguchi (1976). The composition and purity of the extract were assessed by amino acid and hexosamine analyses, infrared spectroscopy and
301
M. BOUVIER et al.
302
cellulose acetate electrophoresis (Schuchman and Desnick, 1981); it contained >95% chondroitin-4sulphate and ~5% protein. The composition and structure of the sponges were characterized by amino acid and hexosamine analyses, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, high-angle X-ray diffraction and differential scanning calorimetry. The collagen sponges were composed of native type I and type III collagen (respectively 90-95% and 4-9% of sponge dry weight) and less than 0.1% (sponge dry weight) of glycosaminoglycans. The sponges had a sulphate collagen
electron
Ultrastructure
microscopy.
of human
At
20
and
50 days of culture, samples were fixed in a 2% glutaraldehyde+cacodylate buffer (0.15 M, pH 7.4) solution at 4°C for 2 h. After rinsing in cacodylate buffer (0.1 M, pH 7.4) for 2 h, samples were postfixed in a 1% osmic acid+acodylate buffer solution at 4’C for 1 h. Specimens were dehydrated in ethanol and embedded in Epon 812. Ultra-thin sections were stained with uranyl acetate and lead citrate and examined in a HU 12 A Hitachi or a 1200 EX JEOL electron microscope. Electron microprobe analysis. X-ray microprobe analysis (CAMEBAX, CAMECA, France) was used to study the qualitative chemical composition of small patches of dense deposits observed in collagenxhondroitin sulphate sponges. Unstained ultrathin sections were placed on aluminium grids. Calcium was detected by energy dispersive spectrometry (very full scale: 120 imp/20 s; absorbed current: R4; accelerating voltage: 45 keV) and phosphorus by wavelength dispersive spectrometry because of the superimposition of the osmium and phosphorus peaks in energy dispersive spectrometry analysis [monocristal: pentaerythritol (2d sin fI = ki, peak:
1 = 0.7065,
background:
I = 0.715)].
Von Kossa staining Control sponges and samples seeded with cells were fixed after 12 and 20 days of culture in a 2% glutaraldehyde
Plate 1 pulp cells cultured on collagen+hondroitin-4-sulphate
sponges.
Fig. 1. Twenty-day-old culture showing polarized nucleus and numerous organelles. Bundles of microfilaments delineate focal adhesion plaques in contact with the sponge (arrows). An unorganized extracellular matrix has been deposited (arrowheads). M, mitochondria; rER, rough endoplasmic reticulum; S, sponge. x 9000 Fig. 2. Twenty-day-old culture showing small needle-like electron-dense crystals among the matrix sponge (arrows). Typical banded collagen fibres are seen (arrowheads). x 20,000
of the
Fig. 3. Fifty-day cultured cells contained a high concentration of filaments; focal adhesion plaques were seen (arrows). Electron-dense deposits (arrowheads) were scattered throughout the sponge fibres (S). M, mitochondria; N, nucleus; G, Golgi apparatus; rER, rough endoplasmic reticulum; C, cilium. x 24,000 Fig. 4. Fifty-day-old Electron-dense
culture. A fibrillar extracellular matrix has been elaborated by the cells (arrowheads). crystals have been deposited in the sponge fibres (arrows). S, sponge. ~43,000
Ultrastructure
Plate 2 of pulp cells cultured
on collagen
sponges
Fig. 6. Twenty-day-old culture showing the appearance of the organelles. N, nucleus; M, G, Golgi apparatus; rER, rough endoplasmic reticulum. x 26,000 Fig. 7. Twenty-day-old culture. Processes rich in intermediate filaments (13 nm in diameter) in close contact with the sponge fibres (arrows). An elaborated matrix of fibrillar material diameter) has been deposited (arrowhead). S, sponge. x40,000 Fig. 8. Fifty-day-old culture. The extracellular matrix is composed of thick fibrils (50 nm in a banded pattern (arrows) surrounded by a faint fibrillar material (20 nm in diameter; x 24,000 Fig. 9. General cytoplasm and
organization of a 50-day-old culture. Note the numerous the newly deposited extracellular material: arrows, thick material; S, sponge fibres. x 20,000
mitochondria; were observed (13-20 nm in diameter) with arrowhead).
filaments inside the cellular fib&; arrowheads, fibrillar
Mineralization of a three-dimensional
Plate 1
collagen matrix
304
M.
BOUVIER
Plate 2
et al.
Mineralization of a three-dimensional
Plate 3
collagen matrix
305
306
M. BOUVIERer al.
-c . c
‘4
50pm t I
100
12
l-5
m
.
Plate 4
Mineralization of a three-dimensional
Ultrastructure of pulp ceils cultured in collagenchondroitin sulphate sponges
At 20 days of culture, the pulp cells had numerous organelles (mitochondria, endoplasmic reticulum) and a polarized nucleus. They appeared to adhere to the sponge fibres through focal adhesion plaques (Plate Fig. 1). A little synthesized matrix was observed. At higher magnification, small needle-like electron-dense crystals were observed in the sponge matrix which was composed of fibrillar material and organized zones or cross-striated collagen fibres (Plate Fig. 2). At 50 days of culture (Plate Fig. 3), the organelles, consisting of mitochondria, rough endoplasmic reticulum, Golgi apparatus and cilia, were surrounded by a large number of filaments distributed throughout the whole cytoplasm. Numerous electron-dense crystals had been deposited in the sponge, but the areas of faint fibrillar extracellular matrix synthesized by the cells were deposit free (Plate Fig. 4). The X-ray microprobe analysis revealed the presence of calcium and phosphorus in the crystals [Text Fig. 5(a), (b)]. Ultrastructure of cells cultured in the collagen sponge At 20 days of culture, the intracytoplasmic pattern of organelles comprised numerous mitochondria, a well-developed Golgi apparatus and rough endoplasmic reticulum filled with a dense material (Plate Fig. 6). An extracellular matrix composed of fine fibrils (13-20 nm in diameter) was observed (Plate Fig. 7). The cells had elongated processes, rich in intermediate filaments (13 nm in diameter), in close contact with the sponge (Plate Fig. 7). By 50 days, the matrix was organized into thick fibrils (50 nm in diameter; Plate Figs 8-9) with a banded pattern (Plate Fig. 8), surrounded 'bya faint fibrillar material (Plate Figs 8-9). The cellular cytoplasm was filled with numerous filaments (Plate Fig. 9). At neither time were any electron-dense crystals seen in the sponges. Ultrastructure of coiztrol samples
incubated
307
(a)
RESULTS
Both types of sponge,
collagen matrix
for 20 days
VFS: 1
120impI20s Al
w
45 keV
(b)
J
Fig. 5. X-ray microanalysis of the needle-like deposits observed in the collagen+zhondroitin sulphate sponges. (a) Energy dispersive spectrometry revealed the presence of calcium. (b) Wavelength dispersive spectrometry showed the presence of phosphorus.
without cells, were free of crystal deposits (Plate Figs 10-l 1). Von Kossa staining
Neither collagen sponges seeded with pulp cells nor controls (sponges incubated without cells) were Von Kossa stained at any time of culture (Plate Fig. 12). At 12 days of culture, some fibres of the collagenchondroitin sulphate sponges were stained (Plate Fig. 13); at higher magnification fine granular deposits were visualized (Plate Fig. 14). At 20 days of
Plate 3 Ultrastructure of control sponges incubated without cells in the culture medium. No mineralization was observed on the structured banded fibrils (arrowheads) of fine filaments without periodicity (arrows) which constitute the structure of both sponges. Fig. 10. Collagen
M.
308
culture, the collagen
BOUVIER et al.
appeared
DlSCUSSION
The three-dimensional matrices of collagen or collagen
to be reached for mineral nucleation (Arsenault and Ottensmeyer, 1984). A specific role for glycosaminoglycans in the differentiation of growing cells cannot be excluded because glycosaminoglycans can maintain the polarized state of cultured odontoblasts (Tziafas et al., 1988). Therefore, the synthesis and deposition of the non-collagenous proteins involved in mineralization might be induced by chondroitin sulphate (Weinstock and Leblond, 1973; Butler, 1984; Andujar and Magloire, 1989). The lack of mineral deposition in our collagen sponges might reflect an undifferentiated or less differentiated state of the cells. Based on the use of selective extracellular matrix components as culture substrates, we conclude that chondroitin-4-sulphate has a role in the differentiation of dental pulp cells, allowing in vitro mineralization. Acknowledgements-We gratefully acknowledge Dr M. Andujar for critical reading of the manuscript, and Mrs F.
Hemming for her help with the grammar. REFERENCES
Andujar M. B. and Magloire H. (1989) Immunoelectron microscopic localization of dentine gamma-carboxyglutamic acid-containing proteins in differentiating rat odontoblasts. Matrix 9, 17-20. Arsenault A. L. and Ottensmeyer F. P. (1984) Visualization of early intramembranous ossification by electron microscopic and spectroscopic imaging. J. Cell Biol. 98, 911-921. Boukari A. and Ruth J. V. (1981) Comportement d’Cbauches dentaires d’embryons de souris in vitro: maintien de la morphologie coronaire, mineralisation. J. Biol. buccale 9, 349-36 I. Bronkers A. L. J. J. (1983) Effect of oxygen tension on matrix formation and mineralization in hamsters molars during development in vitro. J. Biol. buccale 9, 19-207. Burridge K., Molony L. and Kelly T. (1987) Adhesion plaques: sites of transmembrane interaction between the extracellular matrix and the actin cytoskeleton. J. Cell Sci. Suppl. 8, 211-229. Butler W. T. (1984) Matrix macromolecules of bone and dentin. Collagen Rel. Res. 4, 297-307. Doillon C. J. and Silver F. H. (1986) Collaaen-based wound dressing: effects of hyalurdnic acid ani fibronectin on wound healing. Biomaferials 7, 3-8. Doillon C. J., Silver F. H. and Berg R. (1987) Fibroblast growth on a porous collagen sponge containing hyaluronic acid and fibronectinrBiomater& 8, 195-206 Feit J.. Metelova M. and Sindelka 2. (1970) Incornoration of ‘H-thymidine into damaged pulp. J.’ dent. ‘Res. 49, 783-785. Fitzgerald M. (1979) Cellular mechanics of dental bridge repair using XH-thymidine. J. dent. Res. 58(D), 2198-2206. Goldberg M. and Septier D. (1983) Electron microscopic visualization of proteoglycans in rat incisor predentine and dentine with cuprolinic blue. Archs oral Biol. 28, 79-83. Gotoh Y., Saito S. and Sato A. (1979) Synthesis of procollagen by odontogenic cells of rabbit tooth germ. Biochim. biophys. Acta 587, 253-262. Kleinman H. K., Luckenbill-Edds L., Cannon F. W. and Sephel G. C. (1987) Use of extracellular matrix components for cell culture. Analyt. Biochem. 166, 1-13. Laine M. and Thesleff I. (1986) Development of mouse embryonic molars in vitro: an attempt to design defined culture conditions allowing mineralization. J. Biol. buccale 14, 15-23.
Mineralization
of a three-dimensional
Linde A. (1985) Cells and extracellular matrices of the dental pulp. J. dent. Res. 64, 523-529. Magloire H. and Dumont J. (1976) Etude ultrastructurale de cellules uulnaires humaines cultivees “in oitro”. J. Biol. buccale 4, j-20. Magloire H., Joffre A., (Grimaud J. A., Herbage D., Couble M. L.. Chavrier C. and Dumont J. (1981) Synthesis of type I collagen by human odontoblast-like cells in explant culture: light and electron microscope immunotyping. Cell. Molec. Biol. 21, 429435. Magloire H., Hartmann D. J., Couble M. L., Joffre A., Grimaud J. A., Herbage D. and Ville G. (1984) Cytodifferentiation of human dental pulp cells in explant culture. Les colloques de I’INSERM. Tooth morphogenesis and differentiation. INSERM 125, 119-126. Magloire H., Joffre A. and Hartmann D. J. (1988) Localization and synthesis of type III collagen and fibronectin in human reparative dentin. Histochemisfry 8, 14lkl49. Prince C. W.. Rahemtulla F. and Butler W. T. (1983) Metabolism of rat bone proteoglycans in Go. Biochem. J. 216, 5899596. Prince C. W., Rahemt.ulla F. and Butler W. T. (1984) Incorporation of [%]sulphate into glycosaminoglycans by mineralized tissues in oiro. Biochem. J. 224, 941-945. Ruth J. V., Lesot H., Karcher-Djuricic V., Meyer J. M. and Olive M. (1982) Facts and hypotheses concerning the control of odontoblast differentiation. Dtflerenfiation 21, 7-12. Schuchman E. H. and Desnick R. J. (1981) A new continuous, monodimensional electrophoretic system for the separation and quantitation of individual glycosaminoglycans. Analyt. Biochem. 117, 419426. Shuttleworth C. A., Berry L. and Wilson N. (1980) Collagen synthesis in rabbit dental pulp fibroblast cultures. Archs oral Biol. 25, 201-205.
collagen
matrix
309
Takagi M., Parmeley R. T. and Denys F. R. (1981) Ultrastructural localization of complex carbohydrates in odontoblasts, predentin and dentine. J. Histochem. Cytochem. 29, 747-758. Takuma S. and Nagai N. (1971) Ultrastructure of rat odontoblasts in various stages of their development and maturation. Archs oral Biol. 16, 993-1011. Taniguchi N. (1976) Separation and identification of glycosaminoglycans. Sep. Purif. M. 5, 2477299. Thesleff I. (1976) Differentiation of odontogenic tissues in organ culture. &and. J. dent. Res. 84, 353-356. Thesleff I. and Hurmerinta K. (1981) Tissue interactions in tooth development. Differentiation 18, 75-88. Tziafas D., Amar S., Staubli A.. Meyer J. M. and Ruth J. V. (1988) Effects of glycosaminoglycans on in oitro mouse dental cells. Archs oral Biol. 10, 735-740. Weinstock M. and Leblond C. P. (1973) Radioautographic visualization of the deposition of a phosphoprotein at the mineralization front in the dentine of the rat incisor. J. Cell Biol. 56, 838-845. Weinstock M. and Leblond C. C. (1974) Synthesis, migration and release of precursor collagen by odontoblast as visualized by autoradiography after ‘H-proline administration. J. Cell Biol. 60, 922127. Wigglesworth D. J. (1968) Formation and mineralization of enamel and dentine by rat tooth germs in vitro. Expl Cell Res. 49, 21 I-215. Yamada M., Bringas P., Grodin M., MacDougall M., Cumings E., Grimmett J., Weliky B. and Slavkin H. C. (1980) Chemically defined organ culture of embryonic mouse teeth organs: morphogenesis, dentinogenesis and amelogenesis. J. Biol. buccale 8, 127-139. Zusman W. V. and Ioachim H. L. (1964) Growth of odontoblasts in vitro. Dental tissue culture studies. Lab. Itwest. 13, 371-377.
0003-9969/90 $3.00+0.00 Press plc
Printed in Great Britain. All rights reserved
Copyright c 1990Pergamon
IN VITRO MINERALIZATION OF A THREE-,DIMENSIONAL COLLAGEN MATRIX BY HUMAN DENTAL PULP CELLS IN THE PRESENCE OF CHONDROITIN SULPHATE M. BOUVIER,‘,~*A. JOFFRE’ and H. MAGLOIRE’ ‘Laboratoire d’Histophysiologie et de Pathologie des Tissus Dentaires, Rue Guillaume Paradin, 69372, Lyon Cedex 8 and ZBIOETICA S.A., 32 rue Saint Jean De Dieu, 69007, Lyon, France (Received 3 May 1989; accepted 29 September
1989)
Summary-These matrices were used as cell culture substrates to investigate the influence of extracellular molecules on mineralization. Pulp cells seeded in type I collagen or type I collagen-chondroitin-4-sulphate sponges were able to grow and were morphologically similar to cells responsible for reparative dentine formation in tko. In sponges consisting of collagen only, the cells elaborated an abundant new matrix which became organized with time and consisted of collagen fibres surrounded by fibrillar material, but no mineralization was observed. In collagen-chondroitin sulphate sponges, cells deposited less and poorly organized mal.rix; in these, calcification occurred, increasing with time, and at the ultrastructural level, small needle-like crystals containing calcium and phosphorus were scattered throughout the sponge fibres. These observations suggest that chondroitin sulphate might influence in virro calcification induced by pulp cells. Key words: collagen, mineralization, chondroitin sulphate, dental pulp.
INTRODUCTION Dental pulp is a loose mesenchymal tissue, characterized by its particular location, and almost entirely enclosed in a mineralized tissue, dentine. Dentine matrix contains type I collagen associated with noncollagenous proteins (including phosphoproteins and Gla-proteins) and proteoglycans in which the predominant glycosammoglycan in numerous species is chondroitin sulphate (Linde, 1985). Dentine is elaborated by highly differentiated cells, the odontoblasts, located at the periphery of the pulp. Their terminal differentiation occurs in a specific temporo-spatial pattern mediated by epithelial-mesenchymal interactions (Thesleff aml Hurmerinta, 1981; Ruth et al., 1982). When a tooth become carious, a new mineralized scar tissue, reparative dentine, is elaborated by underlying differentiated pulp cells (Feit, Metelova and Sindelka, 1970; Fitzgerald, 1979; Magloire, Joffre and Hartmann, 1988). The origin of this induction is still unclear; epithelial-mesenchymal interactions appear not to be involved as epithelial cells and specific basement membrane components have not been detected. One approach to the study of reparative dentine formation consists of growing pulp cells in tissue culture (Zusman and Ioachim, 1964; Magloire and Dumont, 1976; Got’oh, Saito and Sato, 1979; Shuttleworth, Berry and Wilson, 1980; Magloire et nl., 1981). Partial cytodifferentiation of explanted pulp *Address correspondence to: M. Bouvier, Laboratoire d’Histologie, Facultb d’Odontologie, Rue G. Paradin, 69372, Lyon, Cedex 8, France.
cells has been demonstrated through modulation of protein synthesis (Magloire ef al., 1984), but no mineralization was observed. Further differentiation has been obtained by using components of the extracellular matrix as substrates for the cultured cells (Kleinman et al., 1987). Three-dimensional culture matrices consisting of native type I collagen supplemented with glycosaminoglycans or fibronectin have been shown to control the regulation of tendon and dermal fibroblast metabolism (Doillon and Silver, 1986; Doillon, Silver and Berg, 1987). We have now used threedimensional culture substrates consisting of components analogous to the dentine matrix to study whether human pulpal cells grown on these type I collagen or type I collagen-chondroitin-4-sulphate sponges could induce calcification. MATERIALS AND
METHODS
Collagen sponges were manufactured under patent by BIOETICA (Lyon, France) from the skin of young calves. In brief, the dermis had been separated, ground, extensively washed in phosphate buffer (pH 7.8) and in distilled water, swollen in dilute acetic acid, freeze-dried and reticulated by heat. The samples were sterilized by ethylene oxide. Collagenchondroitin-4-sulphate sponges were prepared as above but with the addition of chondroitin-4-sulphate before freeze-drying. Chondroitin sulphate was extracted from bovine nasal cartilage by alkaline treatment and ethanol precipitation, essentially as described by Taniguchi (1976). The composition and purity of the extract were assessed by amino acid and hexosamine analyses, infrared spectroscopy and
301
M. BOUVIER et al.
302
cellulose acetate electrophoresis (Schuchman and Desnick, 1981); it contained >95% chondroitin-4sulphate and ~5% protein. The composition and structure of the sponges were characterized by amino acid and hexosamine analyses, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, high-angle X-ray diffraction and differential scanning calorimetry. The collagen sponges were composed of native type I and type III collagen (respectively 90-95% and 4-9% of sponge dry weight) and less than 0.1% (sponge dry weight) of glycosaminoglycans. The sponges had a sulphate collagen
electron
Ultrastructure
microscopy.
of human
At
20
and
50 days of culture, samples were fixed in a 2% glutaraldehyde+cacodylate buffer (0.15 M, pH 7.4) solution at 4°C for 2 h. After rinsing in cacodylate buffer (0.1 M, pH 7.4) for 2 h, samples were postfixed in a 1% osmic acid+acodylate buffer solution at 4’C for 1 h. Specimens were dehydrated in ethanol and embedded in Epon 812. Ultra-thin sections were stained with uranyl acetate and lead citrate and examined in a HU 12 A Hitachi or a 1200 EX JEOL electron microscope. Electron microprobe analysis. X-ray microprobe analysis (CAMEBAX, CAMECA, France) was used to study the qualitative chemical composition of small patches of dense deposits observed in collagenxhondroitin sulphate sponges. Unstained ultrathin sections were placed on aluminium grids. Calcium was detected by energy dispersive spectrometry (very full scale: 120 imp/20 s; absorbed current: R4; accelerating voltage: 45 keV) and phosphorus by wavelength dispersive spectrometry because of the superimposition of the osmium and phosphorus peaks in energy dispersive spectrometry analysis [monocristal: pentaerythritol (2d sin fI = ki, peak:
1 = 0.7065,
background:
I = 0.715)].
Von Kossa staining Control sponges and samples seeded with cells were fixed after 12 and 20 days of culture in a 2% glutaraldehyde
Plate 1 pulp cells cultured on collagen+hondroitin-4-sulphate
sponges.
Fig. 1. Twenty-day-old culture showing polarized nucleus and numerous organelles. Bundles of microfilaments delineate focal adhesion plaques in contact with the sponge (arrows). An unorganized extracellular matrix has been deposited (arrowheads). M, mitochondria; rER, rough endoplasmic reticulum; S, sponge. x 9000 Fig. 2. Twenty-day-old culture showing small needle-like electron-dense crystals among the matrix sponge (arrows). Typical banded collagen fibres are seen (arrowheads). x 20,000
of the
Fig. 3. Fifty-day cultured cells contained a high concentration of filaments; focal adhesion plaques were seen (arrows). Electron-dense deposits (arrowheads) were scattered throughout the sponge fibres (S). M, mitochondria; N, nucleus; G, Golgi apparatus; rER, rough endoplasmic reticulum; C, cilium. x 24,000 Fig. 4. Fifty-day-old Electron-dense
culture. A fibrillar extracellular matrix has been elaborated by the cells (arrowheads). crystals have been deposited in the sponge fibres (arrows). S, sponge. ~43,000
Ultrastructure
Plate 2 of pulp cells cultured
on collagen
sponges
Fig. 6. Twenty-day-old culture showing the appearance of the organelles. N, nucleus; M, G, Golgi apparatus; rER, rough endoplasmic reticulum. x 26,000 Fig. 7. Twenty-day-old culture. Processes rich in intermediate filaments (13 nm in diameter) in close contact with the sponge fibres (arrows). An elaborated matrix of fibrillar material diameter) has been deposited (arrowhead). S, sponge. x40,000 Fig. 8. Fifty-day-old culture. The extracellular matrix is composed of thick fibrils (50 nm in a banded pattern (arrows) surrounded by a faint fibrillar material (20 nm in diameter; x 24,000 Fig. 9. General cytoplasm and
organization of a 50-day-old culture. Note the numerous the newly deposited extracellular material: arrows, thick material; S, sponge fibres. x 20,000
mitochondria; were observed (13-20 nm in diameter) with arrowhead).
filaments inside the cellular fib&; arrowheads, fibrillar
Mineralization of a three-dimensional
Plate 1
collagen matrix
304
M.
BOUVIER
Plate 2
et al.
Mineralization of a three-dimensional
Plate 3
collagen matrix
305
306
M. BOUVIERer al.
-c . c
‘4
50pm t I
100
12
l-5
m
.
Plate 4
Mineralization of a three-dimensional
Ultrastructure of pulp ceils cultured in collagenchondroitin sulphate sponges
At 20 days of culture, the pulp cells had numerous organelles (mitochondria, endoplasmic reticulum) and a polarized nucleus. They appeared to adhere to the sponge fibres through focal adhesion plaques (Plate Fig. 1). A little synthesized matrix was observed. At higher magnification, small needle-like electron-dense crystals were observed in the sponge matrix which was composed of fibrillar material and organized zones or cross-striated collagen fibres (Plate Fig. 2). At 50 days of culture (Plate Fig. 3), the organelles, consisting of mitochondria, rough endoplasmic reticulum, Golgi apparatus and cilia, were surrounded by a large number of filaments distributed throughout the whole cytoplasm. Numerous electron-dense crystals had been deposited in the sponge, but the areas of faint fibrillar extracellular matrix synthesized by the cells were deposit free (Plate Fig. 4). The X-ray microprobe analysis revealed the presence of calcium and phosphorus in the crystals [Text Fig. 5(a), (b)]. Ultrastructure of cells cultured in the collagen sponge At 20 days of culture, the intracytoplasmic pattern of organelles comprised numerous mitochondria, a well-developed Golgi apparatus and rough endoplasmic reticulum filled with a dense material (Plate Fig. 6). An extracellular matrix composed of fine fibrils (13-20 nm in diameter) was observed (Plate Fig. 7). The cells had elongated processes, rich in intermediate filaments (13 nm in diameter), in close contact with the sponge (Plate Fig. 7). By 50 days, the matrix was organized into thick fibrils (50 nm in diameter; Plate Figs 8-9) with a banded pattern (Plate Fig. 8), surrounded 'bya faint fibrillar material (Plate Figs 8-9). The cellular cytoplasm was filled with numerous filaments (Plate Fig. 9). At neither time were any electron-dense crystals seen in the sponges. Ultrastructure of coiztrol samples
incubated
307
(a)
RESULTS
Both types of sponge,
collagen matrix
for 20 days
VFS: 1
120impI20s Al
w
45 keV
(b)
J
Fig. 5. X-ray microanalysis of the needle-like deposits observed in the collagen+zhondroitin sulphate sponges. (a) Energy dispersive spectrometry revealed the presence of calcium. (b) Wavelength dispersive spectrometry showed the presence of phosphorus.
without cells, were free of crystal deposits (Plate Figs 10-l 1). Von Kossa staining
Neither collagen sponges seeded with pulp cells nor controls (sponges incubated without cells) were Von Kossa stained at any time of culture (Plate Fig. 12). At 12 days of culture, some fibres of the collagenchondroitin sulphate sponges were stained (Plate Fig. 13); at higher magnification fine granular deposits were visualized (Plate Fig. 14). At 20 days of
Plate 3 Ultrastructure of control sponges incubated without cells in the culture medium. No mineralization was observed on the structured banded fibrils (arrowheads) of fine filaments without periodicity (arrows) which constitute the structure of both sponges. Fig. 10. Collagen
M.
308
culture, the collagen
BOUVIER et al.
appeared
DlSCUSSION
The three-dimensional matrices of collagen or collagen
to be reached for mineral nucleation (Arsenault and Ottensmeyer, 1984). A specific role for glycosaminoglycans in the differentiation of growing cells cannot be excluded because glycosaminoglycans can maintain the polarized state of cultured odontoblasts (Tziafas et al., 1988). Therefore, the synthesis and deposition of the non-collagenous proteins involved in mineralization might be induced by chondroitin sulphate (Weinstock and Leblond, 1973; Butler, 1984; Andujar and Magloire, 1989). The lack of mineral deposition in our collagen sponges might reflect an undifferentiated or less differentiated state of the cells. Based on the use of selective extracellular matrix components as culture substrates, we conclude that chondroitin-4-sulphate has a role in the differentiation of dental pulp cells, allowing in vitro mineralization. Acknowledgements-We gratefully acknowledge Dr M. Andujar for critical reading of the manuscript, and Mrs F.
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