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Human ameloblastomas in vitro: Light microscopical and ultrastructural observations
British Journal of Oral and Maxillofacial Surgery (1985) 23, 326332 0 1985 The British Association of Oral and Maxillofacial Surgeons
HUMAN
AMELOBLASTOMAS
IN VITRO:
ULTRASTRUCTURAL G.
STENMAN,
The Departments
D.D.s.,
LIGHT
MICROSCOPICAL
AND
OBSERVATIONS
Ph.D.‘. 2 J. LILJA, D.D.s., S. SAGNE, D.D.s., Ph.D.'
M.D.,
Ph.D.l
and
of Oral Pathology’ and Oral Surgery’, Faculty of Odontology, University of Giiteborg, Giiteborg, Sweden.
Summary. The light microscopical and ultrastructural morphology in vitro of one case each of human plexiform, follicular and acanthomatous ameloblastoma are described. Ultrastructural analysis of cultured tumour cells revealed that the main cell type growing in vitro displayed morphological features typical of columnar cells or preameloblast-like cells of ameloblastomas. Irrespective of histological type all cases showed indistinguishable in vitro light microscopical and ultrastructural morphology. These findings strongly suggest that the columnar cell type accounts for the main proliferative capacity of ameloblastomas in vitro and most likely also in vivo.
Introduction
During the last decade tissue culture methods have been used extensively in tumour biology for studies of e.g., growth control, cell differentiation and chromosomes. However, to our knowledge, no suitable in vitro system for such studies on odontogenic tumours has been reported. Recently, an in vitro system for cytogenetic studies on human salivary gland neoplasms was developed (Mark et al., 1980). So far, results from banding analyses are available from 81 benign pleomorphic adenomas (Mark et al., 1983; Stenman et al., 1984). These results prompted us to initiate similar in vitro studies on other benign neoplasms, including different types of odontogenic tumours. Among the odontogenic tumours the ameloblastoma is of particular interest due to its locally aggressive behaviour and high recurrence rate. The aim of this investigation was to describe the light microscopical and ultrastructural morphology of three cases of ameloblastomas in vitro. Materials Tumour
and Method
material
(Case 1). A 16-year-old female presented with a cystic destruction in the left side of the body and ramus of the mandible including the second and third molar teeth (Figs. 1 & 2). After biopsy, total extirpation of the lesion was performed. Histopathological examination of both specimens showed a mural, plexiform ameloblastoma (Fig 3). There were no signs of local recurrence 3 years postoperatively. (Case 2). A 73-year-old male presented with a progressive swelling of the left side of the mandible. X-ray examination showed a large cyst-like destruction (Received
24 August
1984; accepted 30 September
Request for reprints to: Dr G. Stenman, Dept of Oral Pathology, of GBteborg, Box 33070, S-400 33 Gijteborg, Sweden.
326
Faculty
1984) of Odontology,
University
HUMAN
Fig.
AMELOBLASTOMAS
IN
1
VITRO
327
Fig. 2
Figure l-Case 1. X-ray showing large cystic destruction in the body and ramus of the mandihle including the second and third molar teeth. Figure 2-Case 1. X-ray, 3 years postoperatively, showing no signs ol recurrence.
extending up into the ramus. Histopathological examination of the totally removed lesion showed a mural, follicular ameloblastoma (Fig. 4). There were no signs of local recurrence 2 years postoperatively. (Case 3). A 49-year-old male presented with an expansion of the right maxillary tuberosity. X-ray examination revealed a destruction of the tuberosity including the lower wall of the maxillary sinus. After a biopsy a block resection of the posterior part of the right maxilla was performed. Histopathological examination of both specimens revealed an acanthomatous ameloblastoma (Fig. 5). There were no signs of local recurrence 2 years postoperatively.
Tissue culture Immediately after surgery fresh unfixed tumour tissue was cut aseptically into small pieces of approximately l-2 cu.mm and was explanted into 50 ml plastic tissue culture bottles. The explants were allowed to adhere to the bottom of the bottles for 30 min, after which 5 ml of Eagle’s Alfa MEM supplemented with 10 per cent foetal calf serum, 1 per cent 200 mM L-Glutamine, 200 units of Benzylpenicillin K/ml and 50 ng of streptomycin/ml were added. The culture bottles were incubated at 37°C in a humidified atmosphere containing 5 per cent CO? in air. The culture medium was changed three times a week. The cultures were examined daily under an inverted phase contrast microscope (Leitz Diavert). Confluent cultures were subcultivated 1:2 after treatment with 0.02 per cent Versene solution and 0.25 per cent Trypsin solution
Histopathology
and electronmicroscopy
The main tumours
were fixed in 4% neutral
buffered
formaldehyde,
embedded
328
BRITISH
JOURNAL
Fig.
OF ORAL
& MAXILLOFACIAL
3
SURGERY
Fig. 4
Fig. 5 Figure Figure Figure
S-Case G-Case S-Case
I. Photomicrograph 2. Photomicrograph 3. Photomicrograph
showing showing showing
the picture of a plexiform ameloblastoma, H&E, the picture of a follicular ameloblastoma. H&E, the picture of an acanthomatous ameloblastoma. x 12s.
X 125. x 125. H&E.
HUMAN
AMELOBLASTOMAS
IN
VITRO
329
in paraffin, sectioned and stained with haematoxylin and eosin and with haematoxylin-van Gieson technique. Primary cultures were fixed in 3.5 per cent glutaraldehyde buffered with 0.075 M cacodylate (pH 7.2) for 2 h at room temperature. Postfixation was carried out in 1 per cent 0~0~ buffered with 0.2 M cacodylate (pH 7.2) for 2 h at +4”C. After dehydration in increasing concentrations of ethanol the specimens were embedded in Epon 812. Ultrathin sectioning was carried out with an Ultrotome III (LKB. Sweden) and staining was performed in a saturated solution of uranylacetate in 30 per cent ethanol and in lead citrate according to Venable and Coggeshall (1965).
Results
Light microscopical
observations
Successful cultures were established from all three ameloblastomas. Irrespective of histological type, all tumours showed similar in vitro growth characteristics. Within 2 to 4 days after the explantation, outgrowth of cells was observed from most explants. It should be noted that the outgrowth from all three tumours was slow. Morphologically, the cultured cells clearly resembled epithelial cells. They had a polygonal or cuboidal shape with a few cytoplasmic extensions. The cells were mostly densely packed and demonstrated typical intercellular bridges. The cytoplasm was slightly granular and contained occasional vacuoles. The often eccentric nuclei were regular and showed a coarse chromatin pattern. Usually only one nucleolus was present. Within the first 2 weeks the mitotic index was low in all cultures. However, from the third week of culture the growth accelerated and mitotic figures became more frequent. After about 6 weeks the cultures were confluent. In most cultures, especially in those from case 3, fibroblast-like cells appeared during the fourth week of culture. The number of these cells increased slowly during subsequent weeks. In primary cultures their frequency never exceeded 5-10 per cent of the total cell population. The first subcultures were made after 6 weeks and thereafter every 2 weeks. After 3 to 4 generations the frequency of fibroblast-like cells increased dramatically and after 6 to 8 generations the cultures consisted mainly of fibroblast-like cells. Ultrastructural observations All three types of ameloblastomas showed very similar ultrastructural morphology and it was not possible to distinguish ultrastructurally between the different types. The cultured cells demonstrated features typical of epithelial cells. Thus they were attached to each other by desmosomes and in connection to them the cytoplasm was slightly condensed (Figs. 6 & 7). In many of the cells there were also numerous bundles of tonofilaments (Fig. 7). Furthermore, the cultured tumour cells had long slender nuclei. However, there was no prominent nucleolus and the outline of the nucleus was rather regular (Fig. 8). Mitochondria were common and were scattered throughout the cells. Within the cells there were also clusters of ribosomes and arrays of rough endoplasmic reticulum (Fig. 9). The Golgi complex was rather well developed with many flattened cisternae gradually transforming into electronlucent vacuoles of diverse form (Fig. 10). Vacuoles containing myelin figures also were observed (cf Fig. 6).
Fig.
kg.
6
7
Figure &-Cultured tumour cells. Desmosome, arrays of rough endoplasmic reticulum and vacuoles containing myelin figures can be seen. Figure 7-Cultured tumour cells. Desmosome with condensation of the cytoplasm and bundles of tonofilaments.
Fig.
8
Fig.
10
Figure &-Cultured tumour cells. Long slender nucleus with regular outline is present. The outline of the cells contains many cytoplasmic extensions. Figure 9-Cultured tumour cells. Mitochondriae and clusters of ribosomes can be seen. Figure IO-Cultured tumour cell. Golgi complex with many flattened cisternae gradually transforming into electron-lucent vacuoles.
HUMAN
AMELOBLASTOMAS
IN
VITRO
331
Discussion
This report describes growth of human ameloblastomas in vitro. By ultrastructural analysis we have demonstrated that the main cell type growing in this in vitro system is an epithelial cell with characteristics of ameloblastoma tumour cells. Information in the literature on cultured ameloblastomas is quite sparse. Heritier et al. (1973) described the establishment of cell cultures from a follicular ameloblastoma of the maxilla. However, the identification of the cultured cells as true tumour cells seemed not to be convincing. The origin of the cultured cells in the present three ameloblastomas has been confirmed by ultrastructural analysis. The findings of desmosomes and bundles of tonofilaments in cultured cells from the three ameloblastomas indicate that the cells are of epithelial origin. The cytoplasmic organelles of the cultured tumour cells were scattered throughout the cytoplasm and showed no signs of being segregated and the Golgi complexes revealed a large number of electron-lucent vacuoles. These features are described as being characteristic of columnar cells (ameloblast-like or preameloblast-like cells) of ameloblastomas (Kim et al., 1979). Irrespective of histological type the cultured cells of all three cases displayed characteristics typical of the columnar cell type. However, the cultured cells differed morphologically slightly from the columnar cells as they had no irregular outline to their nuclei and no prominent nucleoli. These changes might be a result of adaptation to the in vitro environment. The cultured cells showed less resemblance to stellate reticulum cells or squamous cells of ameloblastomas. These findings strongly suggest that the columnar cell type, at least in vitro, accounts for the main proliferative capacity of ameloblastomas. Most likely, this is valid also in vivo. According to this assumption stellate reticulum cells of ameloblastomas might be interpreted as a differentiated line of columnar cells, which has lost its mitotic capacity. Tonofilaments or intermediate filaments of the prekeratin type were, surprisingly, not found in all of the cultured ameloblastoma cells. This finding indicates that epithelial cells when grown in vitro may, at least partly, lose their prekeratin filaments. This assumption is in line with recent findings obtained from in vitro transformed rabbit urothelium (Summerhays et al., 1981) as well as from cultured human pleomorphic adenomas (Rozell et al., 1985). When culturing epithelial tumours in vitro the appearance of stromal cells with increasing time of culture presents a problem. In the present cases of ameloblastomas, the frequency of fibroblast-like cells increased dramatically after 3 to 4 generations in vitro and after 6 to 8 generations most cultures consisted mainly of fibroblast-like cells. By using feeder-layer techniques and/or mitogens promoting growth of epithelial cells it might, however, be possible to maintain epithelial tumours like ameloblastomas in vitro during longer periods. Although the follow-up period of the three ameloblastomas is still rather short, the absence of recurrences suggests a clinically benign behaviour of the lesions. This may, at least partly, be explained by the fact that the mural type of ameloblastomas seem to exhibit a less aggressive clinical behaviour than the solid type of ameloblastomas (Rapidis et al., 1982). Further in vitro analyses might be of help in order to study the factors governing the different clinical behaviours of these two types of lesions. In conclusion the results of this investigation demonstrate that human ameloblastomas can be grown in vitro and that, irrespective of histological type, the cells growing in vitro show ultrastructural features typical of the columnar cell type of
332
BRITISH
JOURNAL
OF ORAL
& MAXILLOFACIAL
SURGERY
ameloblastomas. Furthermore, the results indicate that the present in vitro system might be suitable for cytogenetic analyses of ameloblastomas. Such studies are now in progress. Acknowledgement This investigation
was supported
by grants
from
the Dental
Society
of GBteborg.
References Htritier, M., Jacqueloot, N. & Deminatti, M. (1973). Etyde histochimique, cytogCnCtique et en culture Acrualities odonto-sromatoglogiques, 101, 193. cellulaire d’un amtloblastome maxillaire. Kim, S. K., Nasjleti, C. E. & Weatherbee. L. (1979). F‘me structure of cell types in an ameloblastoma. Journal of Oral Pathology, 8, 319. Mark, J., Dahlenfors, R., Ekedahl, C. & Stenman, G. (1980). The mixed salivary gland tumour-a normally benign human neoplasm frequently showing specific chromosomal abnormalities. Cancer Genetics and Cytogenetics, 2, 231. Mark, J., Dahlenfors, R. & Ekedahl. C. (1983). Cytogenetics of the benign human mixed salivary gland Hereditas, 99, 115. tumour. Rapidis, A. D., Angelopoulos, A. P., Skouteris, C. A. & Papanicolaou, S, (1982). Mural (intracystic) ameloblastoma. International Journul of Oral Surgery, 11, 166. Rozell, B., Stenman, G., Hansson. H.-A., Hansson, G. & Mark, J. (1985). Intermediate filaments in cultured human pleomorphic adenomas. An immunohistochemical study. Acfa Pathologica et Microbiologica Scandinavica Section A. In press. Stenman, G., Mark, J. & Ekedahl, C. (1984). Relationships between chromosomal patterns and protooncogenes in human benign mixed salivary gland tumours. Tumorbiology, 5, 15. Summerhays, I. C., Cheng, Y. S. E., Sun, T.-T. & Chen, L. B. (1981). Expression of keratin and vimentin intermediate filaments in rabbit epithelial cells at different stages of benzo(a)pyrcne induced neoplastic progression. Journal of Cell biology, 90, 63. Venable, J. H. & Coggeshall. R. A. (1965). A simplified lead citrate stain for use in electron microscopy. Journal of Cell Biology, 25, 407.
HUMAN
AMELOBLASTOMAS
IN VITRO:
ULTRASTRUCTURAL G.
STENMAN,
The Departments
D.D.s.,
LIGHT
MICROSCOPICAL
AND
OBSERVATIONS
Ph.D.‘. 2 J. LILJA, D.D.s., S. SAGNE, D.D.s., Ph.D.'
M.D.,
Ph.D.l
and
of Oral Pathology’ and Oral Surgery’, Faculty of Odontology, University of Giiteborg, Giiteborg, Sweden.
Summary. The light microscopical and ultrastructural morphology in vitro of one case each of human plexiform, follicular and acanthomatous ameloblastoma are described. Ultrastructural analysis of cultured tumour cells revealed that the main cell type growing in vitro displayed morphological features typical of columnar cells or preameloblast-like cells of ameloblastomas. Irrespective of histological type all cases showed indistinguishable in vitro light microscopical and ultrastructural morphology. These findings strongly suggest that the columnar cell type accounts for the main proliferative capacity of ameloblastomas in vitro and most likely also in vivo.
Introduction
During the last decade tissue culture methods have been used extensively in tumour biology for studies of e.g., growth control, cell differentiation and chromosomes. However, to our knowledge, no suitable in vitro system for such studies on odontogenic tumours has been reported. Recently, an in vitro system for cytogenetic studies on human salivary gland neoplasms was developed (Mark et al., 1980). So far, results from banding analyses are available from 81 benign pleomorphic adenomas (Mark et al., 1983; Stenman et al., 1984). These results prompted us to initiate similar in vitro studies on other benign neoplasms, including different types of odontogenic tumours. Among the odontogenic tumours the ameloblastoma is of particular interest due to its locally aggressive behaviour and high recurrence rate. The aim of this investigation was to describe the light microscopical and ultrastructural morphology of three cases of ameloblastomas in vitro. Materials Tumour
and Method
material
(Case 1). A 16-year-old female presented with a cystic destruction in the left side of the body and ramus of the mandible including the second and third molar teeth (Figs. 1 & 2). After biopsy, total extirpation of the lesion was performed. Histopathological examination of both specimens showed a mural, plexiform ameloblastoma (Fig 3). There were no signs of local recurrence 3 years postoperatively. (Case 2). A 73-year-old male presented with a progressive swelling of the left side of the mandible. X-ray examination showed a large cyst-like destruction (Received
24 August
1984; accepted 30 September
Request for reprints to: Dr G. Stenman, Dept of Oral Pathology, of GBteborg, Box 33070, S-400 33 Gijteborg, Sweden.
326
Faculty
1984) of Odontology,
University
HUMAN
Fig.
AMELOBLASTOMAS
IN
1
VITRO
327
Fig. 2
Figure l-Case 1. X-ray showing large cystic destruction in the body and ramus of the mandihle including the second and third molar teeth. Figure 2-Case 1. X-ray, 3 years postoperatively, showing no signs ol recurrence.
extending up into the ramus. Histopathological examination of the totally removed lesion showed a mural, follicular ameloblastoma (Fig. 4). There were no signs of local recurrence 2 years postoperatively. (Case 3). A 49-year-old male presented with an expansion of the right maxillary tuberosity. X-ray examination revealed a destruction of the tuberosity including the lower wall of the maxillary sinus. After a biopsy a block resection of the posterior part of the right maxilla was performed. Histopathological examination of both specimens revealed an acanthomatous ameloblastoma (Fig. 5). There were no signs of local recurrence 2 years postoperatively.
Tissue culture Immediately after surgery fresh unfixed tumour tissue was cut aseptically into small pieces of approximately l-2 cu.mm and was explanted into 50 ml plastic tissue culture bottles. The explants were allowed to adhere to the bottom of the bottles for 30 min, after which 5 ml of Eagle’s Alfa MEM supplemented with 10 per cent foetal calf serum, 1 per cent 200 mM L-Glutamine, 200 units of Benzylpenicillin K/ml and 50 ng of streptomycin/ml were added. The culture bottles were incubated at 37°C in a humidified atmosphere containing 5 per cent CO? in air. The culture medium was changed three times a week. The cultures were examined daily under an inverted phase contrast microscope (Leitz Diavert). Confluent cultures were subcultivated 1:2 after treatment with 0.02 per cent Versene solution and 0.25 per cent Trypsin solution
Histopathology
and electronmicroscopy
The main tumours
were fixed in 4% neutral
buffered
formaldehyde,
embedded
328
BRITISH
JOURNAL
Fig.
OF ORAL
& MAXILLOFACIAL
3
SURGERY
Fig. 4
Fig. 5 Figure Figure Figure
S-Case G-Case S-Case
I. Photomicrograph 2. Photomicrograph 3. Photomicrograph
showing showing showing
the picture of a plexiform ameloblastoma, H&E, the picture of a follicular ameloblastoma. H&E, the picture of an acanthomatous ameloblastoma. x 12s.
X 125. x 125. H&E.
HUMAN
AMELOBLASTOMAS
IN
VITRO
329
in paraffin, sectioned and stained with haematoxylin and eosin and with haematoxylin-van Gieson technique. Primary cultures were fixed in 3.5 per cent glutaraldehyde buffered with 0.075 M cacodylate (pH 7.2) for 2 h at room temperature. Postfixation was carried out in 1 per cent 0~0~ buffered with 0.2 M cacodylate (pH 7.2) for 2 h at +4”C. After dehydration in increasing concentrations of ethanol the specimens were embedded in Epon 812. Ultrathin sectioning was carried out with an Ultrotome III (LKB. Sweden) and staining was performed in a saturated solution of uranylacetate in 30 per cent ethanol and in lead citrate according to Venable and Coggeshall (1965).
Results
Light microscopical
observations
Successful cultures were established from all three ameloblastomas. Irrespective of histological type, all tumours showed similar in vitro growth characteristics. Within 2 to 4 days after the explantation, outgrowth of cells was observed from most explants. It should be noted that the outgrowth from all three tumours was slow. Morphologically, the cultured cells clearly resembled epithelial cells. They had a polygonal or cuboidal shape with a few cytoplasmic extensions. The cells were mostly densely packed and demonstrated typical intercellular bridges. The cytoplasm was slightly granular and contained occasional vacuoles. The often eccentric nuclei were regular and showed a coarse chromatin pattern. Usually only one nucleolus was present. Within the first 2 weeks the mitotic index was low in all cultures. However, from the third week of culture the growth accelerated and mitotic figures became more frequent. After about 6 weeks the cultures were confluent. In most cultures, especially in those from case 3, fibroblast-like cells appeared during the fourth week of culture. The number of these cells increased slowly during subsequent weeks. In primary cultures their frequency never exceeded 5-10 per cent of the total cell population. The first subcultures were made after 6 weeks and thereafter every 2 weeks. After 3 to 4 generations the frequency of fibroblast-like cells increased dramatically and after 6 to 8 generations the cultures consisted mainly of fibroblast-like cells. Ultrastructural observations All three types of ameloblastomas showed very similar ultrastructural morphology and it was not possible to distinguish ultrastructurally between the different types. The cultured cells demonstrated features typical of epithelial cells. Thus they were attached to each other by desmosomes and in connection to them the cytoplasm was slightly condensed (Figs. 6 & 7). In many of the cells there were also numerous bundles of tonofilaments (Fig. 7). Furthermore, the cultured tumour cells had long slender nuclei. However, there was no prominent nucleolus and the outline of the nucleus was rather regular (Fig. 8). Mitochondria were common and were scattered throughout the cells. Within the cells there were also clusters of ribosomes and arrays of rough endoplasmic reticulum (Fig. 9). The Golgi complex was rather well developed with many flattened cisternae gradually transforming into electronlucent vacuoles of diverse form (Fig. 10). Vacuoles containing myelin figures also were observed (cf Fig. 6).
Fig.
kg.
6
7
Figure &-Cultured tumour cells. Desmosome, arrays of rough endoplasmic reticulum and vacuoles containing myelin figures can be seen. Figure 7-Cultured tumour cells. Desmosome with condensation of the cytoplasm and bundles of tonofilaments.
Fig.
8
Fig.
10
Figure &-Cultured tumour cells. Long slender nucleus with regular outline is present. The outline of the cells contains many cytoplasmic extensions. Figure 9-Cultured tumour cells. Mitochondriae and clusters of ribosomes can be seen. Figure IO-Cultured tumour cell. Golgi complex with many flattened cisternae gradually transforming into electron-lucent vacuoles.
HUMAN
AMELOBLASTOMAS
IN
VITRO
331
Discussion
This report describes growth of human ameloblastomas in vitro. By ultrastructural analysis we have demonstrated that the main cell type growing in this in vitro system is an epithelial cell with characteristics of ameloblastoma tumour cells. Information in the literature on cultured ameloblastomas is quite sparse. Heritier et al. (1973) described the establishment of cell cultures from a follicular ameloblastoma of the maxilla. However, the identification of the cultured cells as true tumour cells seemed not to be convincing. The origin of the cultured cells in the present three ameloblastomas has been confirmed by ultrastructural analysis. The findings of desmosomes and bundles of tonofilaments in cultured cells from the three ameloblastomas indicate that the cells are of epithelial origin. The cytoplasmic organelles of the cultured tumour cells were scattered throughout the cytoplasm and showed no signs of being segregated and the Golgi complexes revealed a large number of electron-lucent vacuoles. These features are described as being characteristic of columnar cells (ameloblast-like or preameloblast-like cells) of ameloblastomas (Kim et al., 1979). Irrespective of histological type the cultured cells of all three cases displayed characteristics typical of the columnar cell type. However, the cultured cells differed morphologically slightly from the columnar cells as they had no irregular outline to their nuclei and no prominent nucleoli. These changes might be a result of adaptation to the in vitro environment. The cultured cells showed less resemblance to stellate reticulum cells or squamous cells of ameloblastomas. These findings strongly suggest that the columnar cell type, at least in vitro, accounts for the main proliferative capacity of ameloblastomas. Most likely, this is valid also in vivo. According to this assumption stellate reticulum cells of ameloblastomas might be interpreted as a differentiated line of columnar cells, which has lost its mitotic capacity. Tonofilaments or intermediate filaments of the prekeratin type were, surprisingly, not found in all of the cultured ameloblastoma cells. This finding indicates that epithelial cells when grown in vitro may, at least partly, lose their prekeratin filaments. This assumption is in line with recent findings obtained from in vitro transformed rabbit urothelium (Summerhays et al., 1981) as well as from cultured human pleomorphic adenomas (Rozell et al., 1985). When culturing epithelial tumours in vitro the appearance of stromal cells with increasing time of culture presents a problem. In the present cases of ameloblastomas, the frequency of fibroblast-like cells increased dramatically after 3 to 4 generations in vitro and after 6 to 8 generations most cultures consisted mainly of fibroblast-like cells. By using feeder-layer techniques and/or mitogens promoting growth of epithelial cells it might, however, be possible to maintain epithelial tumours like ameloblastomas in vitro during longer periods. Although the follow-up period of the three ameloblastomas is still rather short, the absence of recurrences suggests a clinically benign behaviour of the lesions. This may, at least partly, be explained by the fact that the mural type of ameloblastomas seem to exhibit a less aggressive clinical behaviour than the solid type of ameloblastomas (Rapidis et al., 1982). Further in vitro analyses might be of help in order to study the factors governing the different clinical behaviours of these two types of lesions. In conclusion the results of this investigation demonstrate that human ameloblastomas can be grown in vitro and that, irrespective of histological type, the cells growing in vitro show ultrastructural features typical of the columnar cell type of
332
BRITISH
JOURNAL
OF ORAL
& MAXILLOFACIAL
SURGERY
ameloblastomas. Furthermore, the results indicate that the present in vitro system might be suitable for cytogenetic analyses of ameloblastomas. Such studies are now in progress. Acknowledgement This investigation
was supported
by grants
from
the Dental
Society
of GBteborg.
References Htritier, M., Jacqueloot, N. & Deminatti, M. (1973). Etyde histochimique, cytogCnCtique et en culture Acrualities odonto-sromatoglogiques, 101, 193. cellulaire d’un amtloblastome maxillaire. Kim, S. K., Nasjleti, C. E. & Weatherbee. L. (1979). F‘me structure of cell types in an ameloblastoma. Journal of Oral Pathology, 8, 319. Mark, J., Dahlenfors, R., Ekedahl, C. & Stenman, G. (1980). The mixed salivary gland tumour-a normally benign human neoplasm frequently showing specific chromosomal abnormalities. Cancer Genetics and Cytogenetics, 2, 231. Mark, J., Dahlenfors, R. & Ekedahl. C. (1983). Cytogenetics of the benign human mixed salivary gland Hereditas, 99, 115. tumour. Rapidis, A. D., Angelopoulos, A. P., Skouteris, C. A. & Papanicolaou, S, (1982). Mural (intracystic) ameloblastoma. International Journul of Oral Surgery, 11, 166. Rozell, B., Stenman, G., Hansson. H.-A., Hansson, G. & Mark, J. (1985). Intermediate filaments in cultured human pleomorphic adenomas. An immunohistochemical study. Acfa Pathologica et Microbiologica Scandinavica Section A. In press. Stenman, G., Mark, J. & Ekedahl, C. (1984). Relationships between chromosomal patterns and protooncogenes in human benign mixed salivary gland tumours. Tumorbiology, 5, 15. Summerhays, I. C., Cheng, Y. S. E., Sun, T.-T. & Chen, L. B. (1981). Expression of keratin and vimentin intermediate filaments in rabbit epithelial cells at different stages of benzo(a)pyrcne induced neoplastic progression. Journal of Cell biology, 90, 63. Venable, J. H. & Coggeshall. R. A. (1965). A simplified lead citrate stain for use in electron microscopy. Journal of Cell Biology, 25, 407.