Scand.J. Dent. Res. 1978: 86: 313-324 (Key words: dentinogenesis; incisors; odonloblasts; rat; vinblastine)
Acute and protracted effects of vinblastine on odontoblasts and dentinogenesis in rat incisors HANNE B. MIKKELSEN Anatomy Department C, University of Copenhagen, Copenhagen, Denmark
ABSTRACT - The effects of a large dose of vinblastine sulfate (2 mg/kg body weight) on proliferating odontoblast precursors and secretory odontoblasts in the continuously growing rat incisor were studied. The rats were killed 6 h, 24 h, 3 d and 7 d after vinblastine injection. Most cells in the proliferating zone contained arrested mitoses, or had perished after 24 h. After 3 and 7 d, the odontoblasts derived from this zone were reduced in number, and showed altered cell shapes. The odontoblasts had produced irregular dentin. The secretory odontoblasts had displaced nuclei and altered cell shapes after 24 h. Those most affected were opposite early mineralized dentin. In some incisors the cells had perished. In the protracted experiments almost all the odontoblasts were changed and had produced abnormal dentin. In the early mineralized dentin area, accumulations of cells were present after 3 d, and osteodentin-like material after 7 d. (Accepted for publication 30 May 1978)
Vinblastine sulfate has been reported to inhibit polymerization of labile microtubules by binding to tubulin in the cell (WILSON, CRESWELL &; CHIN 1975). It has been shown that administration of a small dose of vinblastine causes reversible metaphase arrest during cell division (KRISHAN 1968, CREASEY 1975). Larger doses cause functional and morphologic disturbances in differentiated cells (DiEGELMANN
&
PETERKOFSKY
1972,
EHRLICH, ROSS & BORNSTEIN 1974, EKHOLM, ERICSON, JOSEFSSON & MELANDER 1974, ERICSON & LUNDQ,UIST 1975, MoE 1977, MoE & MIKKELSEN
1977). Odontoblast precursors and odontoblasts have been described more recently by TAKUMA (1967) and TAKUMA &;
(1971). TAKUMA ic NAGAI (1971) classified the cells on the lingual side of the rat incisor into marginal dental papilla cells, preodontoblasts, young odontoblasts, old odontoblasts and short odontoblasts. The purpose of this investigation was to study the morphologic changes induced by vinblastine in vivo on the proliferating odontoblast precursors and odontoblasts, ending with odontoblasts which had produced 50-|j.m dentin situated on the labial side of the rat incisor. The protracted effect on the development and function of the various odontoblast stages was also studied. The odontoblast classification made by TAKUMA & NAGAI (1971) was used, and the position of the odontoblast stages was NAGAI
314
MIKKELSEN
correlated with the zones of the odontogenic organ and of the enamel organ, which have recently been described by WARSHAWSKY8C SMITH (1974).
Material and methods Sixteen female Wistar rats weighing 95-135 g were used. Twelve rats received intravenously 2 mg vinblastine sulfate (Velbe®) per kg body weight, administered as a solution of 1 g vinblastine per liter 0.154 M NaCl. Four animals received sham injections of 0.154 M NaCl. The rats were killed by perfusion fixation 6 h, 24 h, 3 d or 7 d after the injections. Each group comprised one control rat and three experimental rats. At sacrifice the animals were anesthetized with Mebumal®. Before the perfusion fixation through the abdominal aorta, 8 ml of a solution of 0.03 M NaNOj in 0.154 M NaCl were infused, while the leh renal vein was cut open. The fixative was 0.3 M glutaraldehyde and 4.5 mM CaClj in 0.1M sodium cacodylate buffer, pH 7.2. The peri usion lasted 30 min. The incisors were removed with surrounding bone, and divided into smaller pieces. The tissues were demineralized for 14 d in a solution of 0.1 M EDTA in 0.2 M glutaraldehyde at 4°C, washed with 0.13 M cacodylate buffer for 2 d, postfixed in a solution of 0.1 M OSO4 in 0.1 M cacodylate buller (or 2 h, and then embedded in Epon®. Some incisors were cut into serial longitudinal sections in the sagittal plane, others, mainly mandibular incisors, were cut into semithin transverse sections at an inten'al of 50 |im and
stained with toluidine blue. For photomicrography a Carl Zeiss photomicroscope, equipped with phase-contrast optics, was used.
Results In the following the odontoblast classification by TAKUMA & NAGAI (1971) will be used, since no significant differences between lingual and labial odontoblasts were observed.
CONTROL RATS
The zone of cells investigated started pulpal to the posterior part of the odontogenic organ and extended, facing the enamel organ, about 2,500 |im incisally. Marginal dental papilla cells (Eigs. 1 and 2) - Apically tbe marginal dental papilla cells were more closely situated than the pulp cells. The cells were star-shaped and varying in size. Incisally, the cells gradually lost their projections and developed into a single layer of cubical and later short columnar cells. The large nuclei were oval with one or two nucleoli. Tbe homogeneous cytoplasm was basophilic, and predentin had not yet
Figs, y-v Control rats. Figs. 6-7. Vinblastine-treated rats. Fig. 1. Star-shaped marginal dental papilla cells (O) facing posterior part ofthe odontogenic organ (OO). x72O. Fig. 2, Cubical marginal dental papilla cells (O) in incisal part of the proliferating zone. M, mitosis. OO, odoniogenic organ. x72O. Fig. 3. Preodontoblasts (O), which have started predentin (P) secretion. Arrow heads, pale central area. OO, odontogenic organ. x72O. Fig. 4. Young odontoblasts (O) lacing dentin (D) and start of enamel secretion zone. OP, odontoblast process. TB, terminal bar. DT, dentinal tubule. P, predentin. E, enamel. x72O. Fig. 5. Old odontoblasts (O). Intercellular space contains blood vessels. OP, odontoblast process. DT, dentinal tubule. P, predentin. D, dentin. x72O. Fig. 6. After 6 h. Young odontoblasts (O) facing start of enamel secretion zone. A pale central area is not apparent. Coarse grains (G) are present in cytoplasm and odontoblast processes (OP) are pale and wide. Dark streaks (DS) are observed intercellularly. DT, dentinal tubule. P, predentin. D, dentin. E, enamel. x72O. Fig. 7. After 24 h. Marginal dental papilla cells (O) in incisal part ol proliferating zone. Numerous arrested mitoses (AM) and dark globules (DG) are observed. 0 0 , odontogenic organ. x450.
EFFEGTS OF VINBLASTINE ON ODONTOBLASTS
315
316
MIKKELSEN
been formed. Mitotic figures occurred pseudostratified cell layer. The tallest both in the dental papilla cell zone and in odontoblasts were about 50-60 (xm in the pulp. The length of the zone was length. The oblong nuclei were situated about 600 |xm. pulpally at different levels. Blood vessels Freodontoblasts (Fig. 3) - The length ofwere observed in the intercellular space the preodontoblast zone was about between the distal part of the 500 (im. It consisted of a single layer of odontoblasts. closely packed low columnar cells, length about 30 (im. The large nuclei were oblong with several nucleoli, and were VINBLASTINE-TREATED RATS generally situated in the pulpal part of the cells. Cytoplasm was basophilic with a The experimental results are summarized pale central area. A thin layer of in Table 1. predentin had been formed. It contained Acute experiments triangular rays of fibers, with the apex After 6 h - The number of marginal dental facing the odontoblasts. Mitotic figures papilla cells containing mitotic figures was were not observed in the odontoblast unchanged or had slightly increased. All layer. mitoses were arrested; the cells were large Young odontoblasts (Fig. 4) - The young and rounded with the chromosomes conodontoblast zone was a layer of closely centrated in the center. Arrested mitoses packed columnar cells, length about were not observed in the preodontoblast 40 (im. The oblong nuclei were situated zone. Some nuclei were displaced to the pulpally, at roughly the same level. center of the cell and a pale central area Cytoplasm was basophilic with a pale was not apparent. In some early young central area. Terminal bars were observed odontoblasts the nuclei were displaced and a and distal to these rather pale odontoblast pale central area was not observed. In the processes continued into the predentin young odontoblasts lying incisal to the and dentin in dentinal tubules. The length start of the enamel secretion zone most of of the zone of young odontoblasts was the cytoplasm was finely grained, but about 900 nm. The ameloblasts had pulpal to the terminal bars course grains started enamel secretion in the center of were found (Fig. 6). The base of the this zone. odontoblast processes and the processes proper were pale and wide. Dark streaks Old odontoblasts (Fig. 5) Old were observed extending from the dentin odontoblasts were organized like a Figs. 8-11. Vinblastine-treated rats. Fig. 8. After 24 h. The young odontoblast zone (O) opposite start of enamel secretion zone. The area is filled with debris (DB) and disorganized cells. In dentin (D) an incremental line (I) is apparent. DG, dark globules. P, predentin. E, enamel. x450. Fig. 9. After 3 d. Rounded preodontoblasts. The nuclei are rounded and predentin is irregular (IP); pulpally a predentin-like substance (PL) is apparent. OO, odontogenic organ. x72O. Fig. 10. After 3 d. Young odontoblasts (O) facing start of enamel secretion zone. The cells are of abnormal shape and contain rounded nuclei. Dentin is irregular (ID), and a predentin-like substance (PL) is observed intercellularly and pulpally. IP, irregular predentin. E, enamel. x72O. Fig. 11. After 3 d. The transitional area between young and old odontoblasts. Distinct difference between odontoblasts and pulp cells is absent; the cells are of all shapes and sizes. In dentin (D) an incremental line (I) is present; newly formed dentin is not apparent. PL, predentin-like substance. P, predentin. E, enamel. M, mitosis. x450.
EFFECTS OF VINBLASTINE ON ODONTOBLASTS
317
318
MIKKELSEN Table 1 The changes in tfie different odontoblast stages
Cell stage
6h
24 h
3d
7d
Marginal dental papilla cells
Arrested mitoses
Most cells arrested mitoses, dark globules
Reduced in number, minor changes
Unaffected
Prcodontobjlasts
Minor changes
Nuclei displaced
Reduced in number Unaffected anomalous shapes
Young odontoCytoplasm blasts (incisal grained to the start ol enamel secretion)
The cells greatly changed or perished
Anomalous shapes 1-2 cell layers
Unaffected
Old odontoblasts Minor changes
Changed shapes
Accumulation of cells, less affected areas
Dentin niches, osteodentin masses, less affected areas
to the intercellular space on a level with the distal part of the cells. Old odontoblasts were normal in shape. The nuclei had various sizes and shapes; the odontoblast processes were very pale.
wide distal part. The nuclei were displaced to the center of the odontoblasts. In the basophilic cytoplasm, a pale central area and terminal bars were less distinct. The odontoblast processes were pale, short and wide. Dentin appeared compact. After 24 h (Fig. 7) - In the marginal dental papilla cell zone the number of cells was Incisal to the start of the enamel secretion reduced. Most cells were rounded and zone, the young odontoblasts were greatly contained arrested mitoses. Large dark changed. Dark globules, distal and pulpal globules were dispersed throughout the to the odontoblasts, were observed in area, some being situated intracellularly. some incisors. In others, the odontoblasts The preodontoblasts were shorter, and were disorganized, and masses of debris numerous nuclei were displaced to the and dark globules were observed (Fig. 8). center of the cells. The homogeneous The subjacent pulp cells were full of cytoplasm was paler in the distal part of debris or had perished. In the dentin of this area a darker stained, incremental the cells. Predentin appeared compact and line was observed running parallel to the often extended into the intercellular space. Young odontoblasts were short with a surface in the predentin-dentin junction. Figs. 12—14. Vinblastine-treated rats. Fig. 12. After 7 d. Early old odontoblasts (O). Small abnormal cells situated in a niche in dentin (D). P, predentin. E, enamel. x450. Fig. 13. After 7 d. Old odontoblasts (O) opposite central part of secretory' ameloblast zone. At left odontoblasts are slightly changed and dentin is irregular (ID). At right odontoblasts are cubical or triangular and irregular dentin is reduced in thickness. PL, predentin-like substance. IP, irregular predentin. E, enamel. x450. Fig. 14. After 7 d. Osteodentin masses (OD) subjacent to transition between abnormal and partly normal enamel (E). Peripheral to irregular predentin (IP) an incremental line (I) is apparent. P, predentin. D, dentin. x380.
EFFECTS OF VINBLASTINE ON ODONTOBLASTS
319
MIKKELSEN
320
Old odontoblasts had anomalous forms. The nuclei were of various shapes and were situated at all levels in the cells. The terminal bars were indistinct and the bases of the odontoblast processes were often wider than normal. The odontoblast processes were pale. Solitary dark globules and some debris were apparent in the intercellular space.
Protracted experiments After 3d
— The youngest marginal dental
papilla cells appeared unaffected. The cubical and columnar dental papilla cells were slightly reduced in number. The rounded or irregular oblong nuclei were orientated in different directions. The cytoplasm was stained inhomogeneously and was rather pale in the distal part of the cell. The preodontoblasts were reduced in number (Fig. 9). Most of the preodontoblasts were short columnar cells, length 15-30 tim; rounded and cubical cells were also present. Predentin, which appeared compact, was of irregular thickness and had ramifications pulpally into the intercellular space. Sometimes a predentin-like substance was observed between the odontoblasts and the pulp cells. Young odontoblasts were affected to various degrees. The early young odontoblasts were reduced in number, and of abnormal shape with a wide distal part (Fig. 10). A few short odontoblast processes were present. Dentin was irregular and dentinal tubules were sparse. The transition between the young odontoblasts and the old odontoblasts was not
distinct. The cells in this transitional zone were of all shapes and sizes and were not aligned. The nuclei were generally round and occupied various positions in the cells. Odontoblast processes and normal dentinal tubules were not apparent. In the
apical part of this zone, the cells lay in one or two layers. A predentin-like substance was situated in the intercellular space and pulpal to the cells. In the incisal part of the transitional zone, the distinct difference between odontoblasts and pulp cells had disappeared (Fig. 11). Accumulations of small abnormal cells of all shapes and sizes were present, and sometimes mitotic figures were observed. The nuclei were of various sizes and were irregularly orientated in the cells. A predentin-like substance was situated between the cells. Dentin appeared normal, but was thinner than the apical, irregular dentin. An incremental line was observed peripheral to the predentindentin junction. It started slightly apical to the transition between the zone containing entirely abnormal enamel production and the zone containing partly normal enamel production (produced before drug exposure), extending incisally in the investigated area. The distance from the start of the secretory ameloblast zone containing entirely abnormal enamel production, to the start of the zone containing partly normal enamel production (produced before drug exposure) was on average 1,350 ^m in the mandibular incisor. A few areas with apparently unaffected odontoblasts were observed in the most incisal part of the investigated area. The cells resembled young odontoblasts and blood vessels were absent from the intercellular space. The dentin appeared normal, apart from the incremental line, and was thicker than the surrounding dentin. After 7 d - Marginal dental papilla cells, preodontoblasts and young odontoblasts appeared unaffected with respect to appearance, number and dentin production. Old odontoblasts exhibited various changes. Apically, the old odontoblasts were almost normal. In a few areas, small
EFFECTS OF VINBLASTINE ON ODONTOBLASTS abnormal cells were situated in niches in the dentin (Fig. 12). More incisally, old odontoblasts, which had irregular nuclei and were of various lengths and widths, were observed; the cells became progressively smaller in number. The dentin was irregular and appeared laminated with sparse dentinal tubules. Subjacent and incisal to the middle of the secretory ameloblast zone, the dentin layer became thinner and more irregular (Fig. 13). On the basis of its appearance, the dentin could be divided into two zones: (1) almost the entire dentin layer was changed; (2) more than lOjim dentin was normal, and in this an incremental line was present. In the first zone, the odontoblasts had various shapes. Some were columnar, others were cubical; in addition, almost triangular cells were observed, and the odontoblasts rarely had lateral contacts. Most nuclei were rounded. In the dentin, which appeared laminated and thinner than normal, only a few dentinal tubules were seen. In the second zone, an incremental line was observed in the normal dentin. It started slightly apical to the transition between the zone containing entirely abnormal enamel production and the zone containing partly normal enamel production (produced before drug exposure), extending incisally in the investigated area. Subjacent to the start of the inct~emental line, the most pronounced changes were observed (Fig. 14). The area was filled with masses of an osteodentinlike substance. This substance contained irregular cells and debris and was sometimes lined with odontoblast-like cells pulpally. Peripherally (towards dentin) small irregular cells were present. Between these and the normal dentin a thin layer of irregular predentin with sparse dentinal tubules was present. More incisally, osteodentin, as described above.
321
was observed. In addition, small abnormal odontoblasts were found, situated subjacent to an irregular dentin in which small cells were trapped. Sometimes the odontoblasts and dentin appeared unaffected, apart from the presence of an incremental line. Discussion
In the acute experiments, the marginal dental papilla cells and the late young odontoblasts were markedly affected. After 6 h, solitary marginal dental papilla cells showed arrested mitoses, and after 24 h, the number of cells was reduced and most cells contained arrested mitoses. Late young odontoblasts had, after 6 h, displaced nuclei and grained cytoplasm. After 24 h, the odontoblasts which were most affected, were disorganized, and masses of debris were present. The remaining stages were changed to some extent, with displaced nuclei and slighdy altered shapes. The acute and protracted effects of vinblastine on ameloblasts have recently been reported (MOE 197 7, MoE 8c MIKKELSEN 1977). In the protracted experiments, ameloblasts, which were secretory at the time of vinblastine administration, had produced normal enamel before drug exposure, and abnormal enamel after. Late differentiating ameloblasts had only produced abnormal enamel. hi the protracted experiments of the present study, it was difficult to evaluate which stages the odontoblasts were in at the time of vinblastine exposure, because of the abnormal appearance of the cells. The ameloblast stages and the migration of ameloblasts and odontoblasts in incisors of untreated rats have recently been described (WARSHAWSKY fc SMITH
MIKKELSEN
322
VINBLASTINE INJECTION MD
PO
YO
AFTER 3 DAYS MD
00 SA PO YO
PA
00 . illiiiliiliB
AFTER 7 DAYS
AE
NE MD
PO
YO 0 0
AE Fig. n. odontoblast stages at time of vinhlastine injection and their migration after 3 and 7 d. AE, abnormal enamel. MD, marginal dental papilla cells. NE, normal enamel. OO, old odontoblasts. PA, post-secretory ameloblasts. PO, preodontoblasts. SA, secretory amelohlasts. YO, young odontoblasts.
1974, SMITH & WARSHAWSKY 1975, 1976). It was reported that during the first 8 d odontoblasts labeled with ^H-thymidine migrate parallel to, and behind, the labeled ameloblasts. Assuming that the individual ameloblasts and odontoblasts were facing each other during the experimental period, it may be possible to determine the odontoblast stages at the dme of drug administration, on the appearance of the ameloblasts. In the present experiment, the secretion and migration of the cells appeared delayed, since the abnormal enamel and dentin, observed in the protracted experiments, were absent in the acute experiments. Furthermore, after 3 d, the distance from the start of the secretory ameloblast zone to the zone containing normal enamel (produced before vinblastine injection) was much shorter than expected on the basis of the cell migration rates described by SMITH &; WARSHAWSKY (1975). Assuming that the individual odontoblasts and ameloblasts were
opposite each other during the experimental period, and that the cell migration was delayed, the old odontoblasts were, after 3 d, situated opposite the center of the secretory ameloblast zone, and after 7 d, at the level of the post-secretory ameloblast zone (see Fig. 15). After 3 d, young odontoblasts were found in the early old odontoblast zone, and after 7 d, opposite the end of the secretory ameloblast zone. Preodontoblasts were found in the transitional zone between young and old odontoblasts after 3 d, and after 7 d, opposite the incisal half of the secretory ameloblast zone. After 3 d, the oldest of the marginal dental papilla cells had become early young odontoblasts, which after 7 d were opposite the central part of the secretory ameloblast zone. The changes observed in the present experiments were less severe in proliferating odontoblasts, but were very severe in differentiated secretory odontoblasts. Thus the marginal dental papilla cells were, in the acute experiments, arrested
EFFECTS OF VINBLASTINE ON ODONTOBLASTS in the metaphase, and had an abnormal appearance after 7 d. This is in accordance with the known effect of vinblastine on microtubules (WILSON et al. 1975), It is known that vinblastine binds to tubulin and inhibits polymerization of mitotic and cytoskeletal microtubules. It has also been suggested that vinblastine exposure, as a secondary effect, may lead to chromosome damage and rupture in dividing cells (SENTEIN 1964), The severe changes observed in differentiated odontoblasts involved disorganization of the cells and the presence of dark globules in the acute experiments; after 7 d, osteodentin and debris were present adjacent to the most differentiated odontoblasts. These changes may be due to the fact that the odontoblasts, owing to the absent microtubules, had lost their morphologic and functional polarity, leading to loss of cells. As to the presence of osteodentin, ADKINS (1972) suggested that in injured pulpal tissue, the cells responsible for formation of osteodentin may develop from the undifferentiated cells ofthe pulp. Changes of a similar kind to those of the present study have been reported 2 weeks after exposure to vincristine (STENE & KOPPANG1976).
DiEGELiVIANN,
R,
F,
323
&
PETERKOESKY,
B,:
Inhibition of collagen secretion from bone and cultured fibroblasts by microtubular disruptive drugs, Proc. Natl. Acad. Sei. USA 1972:69:892-896, EHRLICH, H , P,, ROSS, R, & BORNSTEIN, P , :
Effects of antimicrotubular agents on the secretion of collagen, J, Cell Biol. 1974: 62: 390-405. EKHOLM, R,, ERICSON, L, E,,JOSEFSSON, J, O, & MELANDER, A , : In vivo action of vinblastine
on thyroid ultrastructure and hormone secretion. Endocrinology 1974: 94: 641-649, ERICSON, L, E, & LUNDQ.UIST, L : Effect of vinblastine in vivo on ultrastructure and insulin releasing capacity of the B-cell following sulphonylurea and isopropylnoradrenaline, Diabetologia 1975: 11: 467-473, KRISHAN, A , : Time-lapse and ultrastructure studies on the reversal of mitotic arrest induced by vinblastine sulfate in Earle's L cells,
J.
'Natl.
Cancer
Inst.
1968: 4 1 :
581-595, MoE, H,: On the effect of vinblastine on •ameloblasts ot rat incisors in vivo, 3, Acute and protracted effect on differentiating ameloblasts, A light microscopical study, Acta Pathol.
Microbwl.
Scand.
Sect. A. 1 9 7 7 :
85: 330-334, MoE, H, & MiKKELSEN, H,: On the effect of vinblastine on ameloblasts of rat incisors in vivo, 2. Protracted effect on secretory ameloblasts, A light microscopical study, Acta
Pathol.
Microbwl.
Scand.
Sect.
A. 1 9 7 7 :
85: 319-329, SENTEIN, P,: Action de la vincaleucoblastine sur l'oeuf en segmentation et analyse du mecanisme mitotique, C. R. Acad. Sei. Paris 1964: 258:4854-4857, Acknowledgments - The author wishes to thank Dr, H. MOE for interest in this work, and SMITH, C, E, & WARSHAWSKY, H , : Cellular BODiL IvERSEN and K, STUB-CHRISTENSEN for renewal in the enamel organ and the odontoblast layer of the rat incisors as skilled technical assistance. followed by radioautography using 'Hthymidine, Anat. Rec. 1975: 183: 523-562, SMITH, C, E, & WARSHAWSKV, H , : Movement of
References ADKINS, K, F, :
The efiect of actinomycin D on differentiation of odontoblasts in the rat. Arch. Oral Biol. 1972: 17: 323-528, CREASEY, W , A , ; Vinca alkaloids and colchicine. In: SARTORELLI, A, C, & JOHNS, D,
G,
(ed,):
Handbook
of
experimental
pharmacology. Springer, Berlin 670-694, 22
1975, pp.
entire cell populations during renewal of the rat incisor as shown by radioautography after labeling with 'H-thymidine, Am. J. Anat. 1976: 145: 225-260, STENE, T . & KOPPANG, H , : The effect of vincristine on dentinogenesis in the rat incisor,
Scand.
J.
Dent.
Res.
1976: 84:
342-344, TAKUMA, S,: Ultrastructure of dentinogenesis.
MIKKELSEN
324 In:
MILES,
A.
E. W.
(ed.): Structural
and
chemical organization of teeth. Academic Press,
London 1967, pp. 325-370. TAKUMA, S. & NAGAI, N . : Ultrastructure of rat
odontohlasts in various stages of their development and maturation. Arch. Oral Biol. 1971: 16: 993-1011. WARSHAWSKY,
H.
&
SMITH,
C.
E.:
Morphological classification of rat incisor Address: University of Copenhagen Anatomy Department C Universitetsparken 1 DK-2100 Copenhagen O Denmark
ameloblasts. Anat. Rec. 1974: 179: 423-446. WILSON, L., CRESWELL,
K. & CHIN,
D.:
The
mechanism of action of vinblastine. Binding of (acetyl-'H) vinhlastine to embryonic cliick brain tubulin and tuhulin from sea urchin sperm tail outer doublet microtuhules. Biochemistry 1975: 14: 5586-5592.
Acute and protracted effects of vinblastine on odontoblasts and dentinogenesis in rat incisors HANNE B. MIKKELSEN Anatomy Department C, University of Copenhagen, Copenhagen, Denmark
ABSTRACT - The effects of a large dose of vinblastine sulfate (2 mg/kg body weight) on proliferating odontoblast precursors and secretory odontoblasts in the continuously growing rat incisor were studied. The rats were killed 6 h, 24 h, 3 d and 7 d after vinblastine injection. Most cells in the proliferating zone contained arrested mitoses, or had perished after 24 h. After 3 and 7 d, the odontoblasts derived from this zone were reduced in number, and showed altered cell shapes. The odontoblasts had produced irregular dentin. The secretory odontoblasts had displaced nuclei and altered cell shapes after 24 h. Those most affected were opposite early mineralized dentin. In some incisors the cells had perished. In the protracted experiments almost all the odontoblasts were changed and had produced abnormal dentin. In the early mineralized dentin area, accumulations of cells were present after 3 d, and osteodentin-like material after 7 d. (Accepted for publication 30 May 1978)
Vinblastine sulfate has been reported to inhibit polymerization of labile microtubules by binding to tubulin in the cell (WILSON, CRESWELL &; CHIN 1975). It has been shown that administration of a small dose of vinblastine causes reversible metaphase arrest during cell division (KRISHAN 1968, CREASEY 1975). Larger doses cause functional and morphologic disturbances in differentiated cells (DiEGELMANN
&
PETERKOFSKY
1972,
EHRLICH, ROSS & BORNSTEIN 1974, EKHOLM, ERICSON, JOSEFSSON & MELANDER 1974, ERICSON & LUNDQ,UIST 1975, MoE 1977, MoE & MIKKELSEN
1977). Odontoblast precursors and odontoblasts have been described more recently by TAKUMA (1967) and TAKUMA &;
(1971). TAKUMA ic NAGAI (1971) classified the cells on the lingual side of the rat incisor into marginal dental papilla cells, preodontoblasts, young odontoblasts, old odontoblasts and short odontoblasts. The purpose of this investigation was to study the morphologic changes induced by vinblastine in vivo on the proliferating odontoblast precursors and odontoblasts, ending with odontoblasts which had produced 50-|j.m dentin situated on the labial side of the rat incisor. The protracted effect on the development and function of the various odontoblast stages was also studied. The odontoblast classification made by TAKUMA & NAGAI (1971) was used, and the position of the odontoblast stages was NAGAI
314
MIKKELSEN
correlated with the zones of the odontogenic organ and of the enamel organ, which have recently been described by WARSHAWSKY8C SMITH (1974).
Material and methods Sixteen female Wistar rats weighing 95-135 g were used. Twelve rats received intravenously 2 mg vinblastine sulfate (Velbe®) per kg body weight, administered as a solution of 1 g vinblastine per liter 0.154 M NaCl. Four animals received sham injections of 0.154 M NaCl. The rats were killed by perfusion fixation 6 h, 24 h, 3 d or 7 d after the injections. Each group comprised one control rat and three experimental rats. At sacrifice the animals were anesthetized with Mebumal®. Before the perfusion fixation through the abdominal aorta, 8 ml of a solution of 0.03 M NaNOj in 0.154 M NaCl were infused, while the leh renal vein was cut open. The fixative was 0.3 M glutaraldehyde and 4.5 mM CaClj in 0.1M sodium cacodylate buffer, pH 7.2. The peri usion lasted 30 min. The incisors were removed with surrounding bone, and divided into smaller pieces. The tissues were demineralized for 14 d in a solution of 0.1 M EDTA in 0.2 M glutaraldehyde at 4°C, washed with 0.13 M cacodylate buffer for 2 d, postfixed in a solution of 0.1 M OSO4 in 0.1 M cacodylate buller (or 2 h, and then embedded in Epon®. Some incisors were cut into serial longitudinal sections in the sagittal plane, others, mainly mandibular incisors, were cut into semithin transverse sections at an inten'al of 50 |im and
stained with toluidine blue. For photomicrography a Carl Zeiss photomicroscope, equipped with phase-contrast optics, was used.
Results In the following the odontoblast classification by TAKUMA & NAGAI (1971) will be used, since no significant differences between lingual and labial odontoblasts were observed.
CONTROL RATS
The zone of cells investigated started pulpal to the posterior part of the odontogenic organ and extended, facing the enamel organ, about 2,500 |im incisally. Marginal dental papilla cells (Eigs. 1 and 2) - Apically tbe marginal dental papilla cells were more closely situated than the pulp cells. The cells were star-shaped and varying in size. Incisally, the cells gradually lost their projections and developed into a single layer of cubical and later short columnar cells. The large nuclei were oval with one or two nucleoli. Tbe homogeneous cytoplasm was basophilic, and predentin had not yet
Figs, y-v Control rats. Figs. 6-7. Vinblastine-treated rats. Fig. 1. Star-shaped marginal dental papilla cells (O) facing posterior part ofthe odontogenic organ (OO). x72O. Fig. 2, Cubical marginal dental papilla cells (O) in incisal part of the proliferating zone. M, mitosis. OO, odoniogenic organ. x72O. Fig. 3. Preodontoblasts (O), which have started predentin (P) secretion. Arrow heads, pale central area. OO, odontogenic organ. x72O. Fig. 4. Young odontoblasts (O) lacing dentin (D) and start of enamel secretion zone. OP, odontoblast process. TB, terminal bar. DT, dentinal tubule. P, predentin. E, enamel. x72O. Fig. 5. Old odontoblasts (O). Intercellular space contains blood vessels. OP, odontoblast process. DT, dentinal tubule. P, predentin. D, dentin. x72O. Fig. 6. After 6 h. Young odontoblasts (O) facing start of enamel secretion zone. A pale central area is not apparent. Coarse grains (G) are present in cytoplasm and odontoblast processes (OP) are pale and wide. Dark streaks (DS) are observed intercellularly. DT, dentinal tubule. P, predentin. D, dentin. E, enamel. x72O. Fig. 7. After 24 h. Marginal dental papilla cells (O) in incisal part ol proliferating zone. Numerous arrested mitoses (AM) and dark globules (DG) are observed. 0 0 , odontogenic organ. x450.
EFFEGTS OF VINBLASTINE ON ODONTOBLASTS
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316
MIKKELSEN
been formed. Mitotic figures occurred pseudostratified cell layer. The tallest both in the dental papilla cell zone and in odontoblasts were about 50-60 (xm in the pulp. The length of the zone was length. The oblong nuclei were situated about 600 |xm. pulpally at different levels. Blood vessels Freodontoblasts (Fig. 3) - The length ofwere observed in the intercellular space the preodontoblast zone was about between the distal part of the 500 (im. It consisted of a single layer of odontoblasts. closely packed low columnar cells, length about 30 (im. The large nuclei were oblong with several nucleoli, and were VINBLASTINE-TREATED RATS generally situated in the pulpal part of the cells. Cytoplasm was basophilic with a The experimental results are summarized pale central area. A thin layer of in Table 1. predentin had been formed. It contained Acute experiments triangular rays of fibers, with the apex After 6 h - The number of marginal dental facing the odontoblasts. Mitotic figures papilla cells containing mitotic figures was were not observed in the odontoblast unchanged or had slightly increased. All layer. mitoses were arrested; the cells were large Young odontoblasts (Fig. 4) - The young and rounded with the chromosomes conodontoblast zone was a layer of closely centrated in the center. Arrested mitoses packed columnar cells, length about were not observed in the preodontoblast 40 (im. The oblong nuclei were situated zone. Some nuclei were displaced to the pulpally, at roughly the same level. center of the cell and a pale central area Cytoplasm was basophilic with a pale was not apparent. In some early young central area. Terminal bars were observed odontoblasts the nuclei were displaced and a and distal to these rather pale odontoblast pale central area was not observed. In the processes continued into the predentin young odontoblasts lying incisal to the and dentin in dentinal tubules. The length start of the enamel secretion zone most of of the zone of young odontoblasts was the cytoplasm was finely grained, but about 900 nm. The ameloblasts had pulpal to the terminal bars course grains started enamel secretion in the center of were found (Fig. 6). The base of the this zone. odontoblast processes and the processes proper were pale and wide. Dark streaks Old odontoblasts (Fig. 5) Old were observed extending from the dentin odontoblasts were organized like a Figs. 8-11. Vinblastine-treated rats. Fig. 8. After 24 h. The young odontoblast zone (O) opposite start of enamel secretion zone. The area is filled with debris (DB) and disorganized cells. In dentin (D) an incremental line (I) is apparent. DG, dark globules. P, predentin. E, enamel. x450. Fig. 9. After 3 d. Rounded preodontoblasts. The nuclei are rounded and predentin is irregular (IP); pulpally a predentin-like substance (PL) is apparent. OO, odontogenic organ. x72O. Fig. 10. After 3 d. Young odontoblasts (O) facing start of enamel secretion zone. The cells are of abnormal shape and contain rounded nuclei. Dentin is irregular (ID), and a predentin-like substance (PL) is observed intercellularly and pulpally. IP, irregular predentin. E, enamel. x72O. Fig. 11. After 3 d. The transitional area between young and old odontoblasts. Distinct difference between odontoblasts and pulp cells is absent; the cells are of all shapes and sizes. In dentin (D) an incremental line (I) is present; newly formed dentin is not apparent. PL, predentin-like substance. P, predentin. E, enamel. M, mitosis. x450.
EFFECTS OF VINBLASTINE ON ODONTOBLASTS
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MIKKELSEN Table 1 The changes in tfie different odontoblast stages
Cell stage
6h
24 h
3d
7d
Marginal dental papilla cells
Arrested mitoses
Most cells arrested mitoses, dark globules
Reduced in number, minor changes
Unaffected
Prcodontobjlasts
Minor changes
Nuclei displaced
Reduced in number Unaffected anomalous shapes
Young odontoCytoplasm blasts (incisal grained to the start ol enamel secretion)
The cells greatly changed or perished
Anomalous shapes 1-2 cell layers
Unaffected
Old odontoblasts Minor changes
Changed shapes
Accumulation of cells, less affected areas
Dentin niches, osteodentin masses, less affected areas
to the intercellular space on a level with the distal part of the cells. Old odontoblasts were normal in shape. The nuclei had various sizes and shapes; the odontoblast processes were very pale.
wide distal part. The nuclei were displaced to the center of the odontoblasts. In the basophilic cytoplasm, a pale central area and terminal bars were less distinct. The odontoblast processes were pale, short and wide. Dentin appeared compact. After 24 h (Fig. 7) - In the marginal dental papilla cell zone the number of cells was Incisal to the start of the enamel secretion reduced. Most cells were rounded and zone, the young odontoblasts were greatly contained arrested mitoses. Large dark changed. Dark globules, distal and pulpal globules were dispersed throughout the to the odontoblasts, were observed in area, some being situated intracellularly. some incisors. In others, the odontoblasts The preodontoblasts were shorter, and were disorganized, and masses of debris numerous nuclei were displaced to the and dark globules were observed (Fig. 8). center of the cells. The homogeneous The subjacent pulp cells were full of cytoplasm was paler in the distal part of debris or had perished. In the dentin of this area a darker stained, incremental the cells. Predentin appeared compact and line was observed running parallel to the often extended into the intercellular space. Young odontoblasts were short with a surface in the predentin-dentin junction. Figs. 12—14. Vinblastine-treated rats. Fig. 12. After 7 d. Early old odontoblasts (O). Small abnormal cells situated in a niche in dentin (D). P, predentin. E, enamel. x450. Fig. 13. After 7 d. Old odontoblasts (O) opposite central part of secretory' ameloblast zone. At left odontoblasts are slightly changed and dentin is irregular (ID). At right odontoblasts are cubical or triangular and irregular dentin is reduced in thickness. PL, predentin-like substance. IP, irregular predentin. E, enamel. x450. Fig. 14. After 7 d. Osteodentin masses (OD) subjacent to transition between abnormal and partly normal enamel (E). Peripheral to irregular predentin (IP) an incremental line (I) is apparent. P, predentin. D, dentin. x380.
EFFECTS OF VINBLASTINE ON ODONTOBLASTS
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320
Old odontoblasts had anomalous forms. The nuclei were of various shapes and were situated at all levels in the cells. The terminal bars were indistinct and the bases of the odontoblast processes were often wider than normal. The odontoblast processes were pale. Solitary dark globules and some debris were apparent in the intercellular space.
Protracted experiments After 3d
— The youngest marginal dental
papilla cells appeared unaffected. The cubical and columnar dental papilla cells were slightly reduced in number. The rounded or irregular oblong nuclei were orientated in different directions. The cytoplasm was stained inhomogeneously and was rather pale in the distal part of the cell. The preodontoblasts were reduced in number (Fig. 9). Most of the preodontoblasts were short columnar cells, length 15-30 tim; rounded and cubical cells were also present. Predentin, which appeared compact, was of irregular thickness and had ramifications pulpally into the intercellular space. Sometimes a predentin-like substance was observed between the odontoblasts and the pulp cells. Young odontoblasts were affected to various degrees. The early young odontoblasts were reduced in number, and of abnormal shape with a wide distal part (Fig. 10). A few short odontoblast processes were present. Dentin was irregular and dentinal tubules were sparse. The transition between the young odontoblasts and the old odontoblasts was not
distinct. The cells in this transitional zone were of all shapes and sizes and were not aligned. The nuclei were generally round and occupied various positions in the cells. Odontoblast processes and normal dentinal tubules were not apparent. In the
apical part of this zone, the cells lay in one or two layers. A predentin-like substance was situated in the intercellular space and pulpal to the cells. In the incisal part of the transitional zone, the distinct difference between odontoblasts and pulp cells had disappeared (Fig. 11). Accumulations of small abnormal cells of all shapes and sizes were present, and sometimes mitotic figures were observed. The nuclei were of various sizes and were irregularly orientated in the cells. A predentin-like substance was situated between the cells. Dentin appeared normal, but was thinner than the apical, irregular dentin. An incremental line was observed peripheral to the predentindentin junction. It started slightly apical to the transition between the zone containing entirely abnormal enamel production and the zone containing partly normal enamel production (produced before drug exposure), extending incisally in the investigated area. The distance from the start of the secretory ameloblast zone containing entirely abnormal enamel production, to the start of the zone containing partly normal enamel production (produced before drug exposure) was on average 1,350 ^m in the mandibular incisor. A few areas with apparently unaffected odontoblasts were observed in the most incisal part of the investigated area. The cells resembled young odontoblasts and blood vessels were absent from the intercellular space. The dentin appeared normal, apart from the incremental line, and was thicker than the surrounding dentin. After 7 d - Marginal dental papilla cells, preodontoblasts and young odontoblasts appeared unaffected with respect to appearance, number and dentin production. Old odontoblasts exhibited various changes. Apically, the old odontoblasts were almost normal. In a few areas, small
EFFECTS OF VINBLASTINE ON ODONTOBLASTS abnormal cells were situated in niches in the dentin (Fig. 12). More incisally, old odontoblasts, which had irregular nuclei and were of various lengths and widths, were observed; the cells became progressively smaller in number. The dentin was irregular and appeared laminated with sparse dentinal tubules. Subjacent and incisal to the middle of the secretory ameloblast zone, the dentin layer became thinner and more irregular (Fig. 13). On the basis of its appearance, the dentin could be divided into two zones: (1) almost the entire dentin layer was changed; (2) more than lOjim dentin was normal, and in this an incremental line was present. In the first zone, the odontoblasts had various shapes. Some were columnar, others were cubical; in addition, almost triangular cells were observed, and the odontoblasts rarely had lateral contacts. Most nuclei were rounded. In the dentin, which appeared laminated and thinner than normal, only a few dentinal tubules were seen. In the second zone, an incremental line was observed in the normal dentin. It started slightly apical to the transition between the zone containing entirely abnormal enamel production and the zone containing partly normal enamel production (produced before drug exposure), extending incisally in the investigated area. Subjacent to the start of the inct~emental line, the most pronounced changes were observed (Fig. 14). The area was filled with masses of an osteodentinlike substance. This substance contained irregular cells and debris and was sometimes lined with odontoblast-like cells pulpally. Peripherally (towards dentin) small irregular cells were present. Between these and the normal dentin a thin layer of irregular predentin with sparse dentinal tubules was present. More incisally, osteodentin, as described above.
321
was observed. In addition, small abnormal odontoblasts were found, situated subjacent to an irregular dentin in which small cells were trapped. Sometimes the odontoblasts and dentin appeared unaffected, apart from the presence of an incremental line. Discussion
In the acute experiments, the marginal dental papilla cells and the late young odontoblasts were markedly affected. After 6 h, solitary marginal dental papilla cells showed arrested mitoses, and after 24 h, the number of cells was reduced and most cells contained arrested mitoses. Late young odontoblasts had, after 6 h, displaced nuclei and grained cytoplasm. After 24 h, the odontoblasts which were most affected, were disorganized, and masses of debris were present. The remaining stages were changed to some extent, with displaced nuclei and slighdy altered shapes. The acute and protracted effects of vinblastine on ameloblasts have recently been reported (MOE 197 7, MoE 8c MIKKELSEN 1977). In the protracted experiments, ameloblasts, which were secretory at the time of vinblastine administration, had produced normal enamel before drug exposure, and abnormal enamel after. Late differentiating ameloblasts had only produced abnormal enamel. hi the protracted experiments of the present study, it was difficult to evaluate which stages the odontoblasts were in at the time of vinblastine exposure, because of the abnormal appearance of the cells. The ameloblast stages and the migration of ameloblasts and odontoblasts in incisors of untreated rats have recently been described (WARSHAWSKY fc SMITH
MIKKELSEN
322
VINBLASTINE INJECTION MD
PO
YO
AFTER 3 DAYS MD
00 SA PO YO
PA
00 . illiiiliiliB
AFTER 7 DAYS
AE
NE MD
PO
YO 0 0
AE Fig. n. odontoblast stages at time of vinhlastine injection and their migration after 3 and 7 d. AE, abnormal enamel. MD, marginal dental papilla cells. NE, normal enamel. OO, old odontoblasts. PA, post-secretory ameloblasts. PO, preodontoblasts. SA, secretory amelohlasts. YO, young odontoblasts.
1974, SMITH & WARSHAWSKY 1975, 1976). It was reported that during the first 8 d odontoblasts labeled with ^H-thymidine migrate parallel to, and behind, the labeled ameloblasts. Assuming that the individual ameloblasts and odontoblasts were facing each other during the experimental period, it may be possible to determine the odontoblast stages at the dme of drug administration, on the appearance of the ameloblasts. In the present experiment, the secretion and migration of the cells appeared delayed, since the abnormal enamel and dentin, observed in the protracted experiments, were absent in the acute experiments. Furthermore, after 3 d, the distance from the start of the secretory ameloblast zone to the zone containing normal enamel (produced before vinblastine injection) was much shorter than expected on the basis of the cell migration rates described by SMITH &; WARSHAWSKY (1975). Assuming that the individual odontoblasts and ameloblasts were
opposite each other during the experimental period, and that the cell migration was delayed, the old odontoblasts were, after 3 d, situated opposite the center of the secretory ameloblast zone, and after 7 d, at the level of the post-secretory ameloblast zone (see Fig. 15). After 3 d, young odontoblasts were found in the early old odontoblast zone, and after 7 d, opposite the end of the secretory ameloblast zone. Preodontoblasts were found in the transitional zone between young and old odontoblasts after 3 d, and after 7 d, opposite the incisal half of the secretory ameloblast zone. After 3 d, the oldest of the marginal dental papilla cells had become early young odontoblasts, which after 7 d were opposite the central part of the secretory ameloblast zone. The changes observed in the present experiments were less severe in proliferating odontoblasts, but were very severe in differentiated secretory odontoblasts. Thus the marginal dental papilla cells were, in the acute experiments, arrested
EFFECTS OF VINBLASTINE ON ODONTOBLASTS in the metaphase, and had an abnormal appearance after 7 d. This is in accordance with the known effect of vinblastine on microtubules (WILSON et al. 1975), It is known that vinblastine binds to tubulin and inhibits polymerization of mitotic and cytoskeletal microtubules. It has also been suggested that vinblastine exposure, as a secondary effect, may lead to chromosome damage and rupture in dividing cells (SENTEIN 1964), The severe changes observed in differentiated odontoblasts involved disorganization of the cells and the presence of dark globules in the acute experiments; after 7 d, osteodentin and debris were present adjacent to the most differentiated odontoblasts. These changes may be due to the fact that the odontoblasts, owing to the absent microtubules, had lost their morphologic and functional polarity, leading to loss of cells. As to the presence of osteodentin, ADKINS (1972) suggested that in injured pulpal tissue, the cells responsible for formation of osteodentin may develop from the undifferentiated cells ofthe pulp. Changes of a similar kind to those of the present study have been reported 2 weeks after exposure to vincristine (STENE & KOPPANG1976).
DiEGELiVIANN,
R,
F,
323
&
PETERKOESKY,
B,:
Inhibition of collagen secretion from bone and cultured fibroblasts by microtubular disruptive drugs, Proc. Natl. Acad. Sei. USA 1972:69:892-896, EHRLICH, H , P,, ROSS, R, & BORNSTEIN, P , :
Effects of antimicrotubular agents on the secretion of collagen, J, Cell Biol. 1974: 62: 390-405. EKHOLM, R,, ERICSON, L, E,,JOSEFSSON, J, O, & MELANDER, A , : In vivo action of vinblastine
on thyroid ultrastructure and hormone secretion. Endocrinology 1974: 94: 641-649, ERICSON, L, E, & LUNDQ.UIST, L : Effect of vinblastine in vivo on ultrastructure and insulin releasing capacity of the B-cell following sulphonylurea and isopropylnoradrenaline, Diabetologia 1975: 11: 467-473, KRISHAN, A , : Time-lapse and ultrastructure studies on the reversal of mitotic arrest induced by vinblastine sulfate in Earle's L cells,
J.
'Natl.
Cancer
Inst.
1968: 4 1 :
581-595, MoE, H,: On the effect of vinblastine on •ameloblasts ot rat incisors in vivo, 3, Acute and protracted effect on differentiating ameloblasts, A light microscopical study, Acta Pathol.
Microbwl.
Scand.
Sect. A. 1 9 7 7 :
85: 330-334, MoE, H, & MiKKELSEN, H,: On the effect of vinblastine on ameloblasts of rat incisors in vivo, 2. Protracted effect on secretory ameloblasts, A light microscopical study, Acta
Pathol.
Microbwl.
Scand.
Sect.
A. 1 9 7 7 :
85: 319-329, SENTEIN, P,: Action de la vincaleucoblastine sur l'oeuf en segmentation et analyse du mecanisme mitotique, C. R. Acad. Sei. Paris 1964: 258:4854-4857, Acknowledgments - The author wishes to thank Dr, H. MOE for interest in this work, and SMITH, C, E, & WARSHAWSKY, H , : Cellular BODiL IvERSEN and K, STUB-CHRISTENSEN for renewal in the enamel organ and the odontoblast layer of the rat incisors as skilled technical assistance. followed by radioautography using 'Hthymidine, Anat. Rec. 1975: 183: 523-562, SMITH, C, E, & WARSHAWSKV, H , : Movement of
References ADKINS, K, F, :
The efiect of actinomycin D on differentiation of odontoblasts in the rat. Arch. Oral Biol. 1972: 17: 323-528, CREASEY, W , A , ; Vinca alkaloids and colchicine. In: SARTORELLI, A, C, & JOHNS, D,
G,
(ed,):
Handbook
of
experimental
pharmacology. Springer, Berlin 670-694, 22
1975, pp.
entire cell populations during renewal of the rat incisor as shown by radioautography after labeling with 'H-thymidine, Am. J. Anat. 1976: 145: 225-260, STENE, T . & KOPPANG, H , : The effect of vincristine on dentinogenesis in the rat incisor,
Scand.
J.
Dent.
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1976: 84:
342-344, TAKUMA, S,: Ultrastructure of dentinogenesis.
MIKKELSEN
324 In:
MILES,
A.
E. W.
(ed.): Structural
and
chemical organization of teeth. Academic Press,
London 1967, pp. 325-370. TAKUMA, S. & NAGAI, N . : Ultrastructure of rat
odontohlasts in various stages of their development and maturation. Arch. Oral Biol. 1971: 16: 993-1011. WARSHAWSKY,
H.
&
SMITH,
C.
E.:
Morphological classification of rat incisor Address: University of Copenhagen Anatomy Department C Universitetsparken 1 DK-2100 Copenhagen O Denmark
ameloblasts. Anat. Rec. 1974: 179: 423-446. WILSON, L., CRESWELL,
K. & CHIN,
D.:
The
mechanism of action of vinblastine. Binding of (acetyl-'H) vinhlastine to embryonic cliick brain tubulin and tuhulin from sea urchin sperm tail outer doublet microtuhules. Biochemistry 1975: 14: 5586-5592.
