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Enamel incremental periodicity in the pig-tailed macaque A polychrome fluorescent labeling study of dental hard tissues.

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Enamel Incremental Periodicity in the Pig-Tailed Macaque: A
Polychrome Fluorescent Labeling Study of Dental Hard Tissues
Department of Anthropology, Hunter College, C .U.N.Y.,New York,
New York
Dental development, Circadian periodicity, Peri-
Bromage and Dean originally outlined a nondestructive
method for the study of enamel formation and concluded that early hominids
resembled the extant apes more closely than they did modern humans in their
rates of growth and maturation. The method used assumed that an enamel
circadian rhythmicity was referable to a longer near-weekly period represented by perikymata (periodic surface growth features). This assumption
became a matter of debate and discussion. In this study, developing teeth in
Mucacu nemestrina were labeled with polychrome fluorescent dyes. Examination of the distribution of these dyes in two sectioned teeth provides experimental confirmation of enamel circadian periodicity.
Recent studies on the rate and pattern of
dental development suggest that the growth
and maturation of early hominids were more
similar to the extant apes than to modern
humans (e.g., Beynon and Dean, 1988; Bromage, 1986; Bromage and Dean, 1985; Conroy and Vannier, 1987; Smith, 1986). This
conclusion contrasts with the previous opinion, derived from combined dental developmental pattern and attrition studies, that
early hominids were more hominine in their
development (Mann, 1975).
Bromage and Dean (1985) have described
a nondestructive method for determining the
duration of early hominid enamel formation
on incompletely formed and nonabraded
(i.e., diagenetic abrasion) permanent incisor
crowns. This method depended on the assumptions that 1)enamel “cross striations”
(an internal enamel incremental growth
line) resulted from a circadian variation in
the rate of matrix secretion by enamel forming cells and 2) an average of seven cross
striations accrued between adjacent “striae
of Retzius” (a more pronounced internal
enamel incremental growth line). Bromage
and Dean (1985) further noted that striae of
Retzius regularly emerge on the incisor
enamel surface as “perikymata” (periodic
surface growth features) and that when
these perikymata are counted their numbers
can be used to calibrate crown formation
times, other dental developmental events,
and the ages at death for some early hominid
specimens. Despite substantial circumstantial evidence for enamel circadian rhythmicity (cf. Dean, 1987a1, critics noted a lack of
experimental proof (cf. Bower, 1985, 1987;
Lewin, 1985).
It was circumstantial evidence that led
first Asper in 1916 and then Gysi in 1931 to
hypothesize that cross-striations and daily
enamel increments were related. They observed that for human canines the number of
cross striations between the time of initial
calcification and crown completion corresponded to the number of days required for
canine development. Furthermore, confirmation of this relationship was provided by
Boyde (1964),who counted enamel cross striations in ground sections of teeth from an
immature individual from an archeological
context and calculated the number of days
between birth (neonatal incremental line)
and death of that individual. The calculated
age was consistent with the dental maturation status at the time of death. Dean (1987a)
provides an in depth review.
Mimura (1939) first provided experimental proof of enamel circadian rhythmicity by
labeling developing teeth with injections of
Received March 5, 1990; accepted March 26,1991
lead acetate and sodium fluoride in the
growing dog, rabbit, and pig. The forming
fronts of developing enamel corresponded to
discrete cross striations in ground sections
that were identified by physicochemical
means. Importantly, the numbers of cross
striations between the lead acetate and sodium fluoride lines corresponded to the
number of days between injections. Subsequent work with other animals, including
macaques, further demonstrated this relationship (Okada, 1943).The circadian rhythmicity of dentine was also confirmed (Okada,
1943) and has been correlated with daily
enamel increments (Kawasaki et al., 1980).
Experimental demonstrations of the circadian rhythmicity of enamel and dentine formation have not been undertaken since
these early pioneering works.
The present study is similar to the early
Japanese works cited above, but it differs in
respect to the labeling substances used. This
study was made possible by timed polychrome fluorescent vital labeling studies of
developing fetal and immatureMucuca nemestrina dental hard tissues. While concern
here is mainly to corroborate early studies on
the circadian rhythmicity of enamel formation, attention is also paid to the circadian
rhythmicity of dentine formation.
The present study benefits from work by
L. Newell-Morris (University of Washington) and colleagues who for the first time
labeled mineralizing fetal tissues with trichromatic fluorescent compounds (NewellMorris and Sirianni, 1982). Pregnant pigtailed macaque (M. nemestrinu) female and
infant hard tissues were sequentially labeled at recorded intervals with three fluorescent substances; DCAF (2,4Bis) N,N’Di
(carboxymethyl) aminomethyl fluorescein,
xylenol orange, and minocycline hydrochloride (Newell-Morris and Sirianni, 1982).
Two developing maxillary first permanent
molars were isolated from the jaws of specimens stored in 70% alcohol. The first specimen (specimen 1)is the crown of the upper
first permanent molar of an animal labeled
postnatally at 2 week intervals with minocycline, xylenol orange, and DCAF in that order. The animal was born at 171 + 2 postconception days on February 6, 1978,
received the first label March 22, 1979, the
last label on April 19, and sacrificed 18 days
The second specimen (specimen 2) considered is also the crown of the upper first
permanent molar of a n animal labeled postnatally at 2 week intervals with DCAF, minocycline, xylenol orange, and minocycline
again. The animal was born August 10,1978
(untimed mating), received the first label
January 11,1979, the last label on February
22, and sacrificed 4 days later.
Teeth were dehydrated and defatted by
refluxing for 7-14 days using 1 : 1 chloroform-methanol in a Soxhlet apparatus and
then transferred to a mixture of methylmethacrylate monomer and styrene and
stored a t 4°C. The embedding medium was
changed three times at 24 hour intervals,
and specimens were then transferred to daylight a t 20°C to polymerize the methacrylate.
Polymerized blocks were sectioned with a
low speed diamond saw and each tooth section ground and polished on both sides with
1/10 diamond compound to 80-100 pm. Fluorochromes were examined with a Zeiss Photomicroscope 1 using incident ultraviolet
light to reveal fluorescent lines denoting the
mineralizing fronts of enamel and dentine a t
the times of administration of the vital labels.
Images were focused until the greatest
optical contrast could be qualitatively ascertained for incremental features and until the
most discrete line could be achieved for fluorescent labels. The planes of focus for corresponding transmitted light and fluorescent
images were not necessarily precisely the
same (illustrating the importance of future
studies by Tandem Scanning Reflected
Light Microscopy of single and very thin
planes of focus a t the limits of optical resolution deep to dental hard tissue (e.g., Boyde
et al., 1983).
Enamel and dentine incremental features
were measured with a video measurement
system attached to the Photomicroscope 1
and calibrated to 1.0 p,m resolution (Via-100,
Boeckeler Instruments, Inc.).
Figure 1 portrays a low magnification image of the buccal-lingual section of specimen
1, illustrating a section through the distallingual cusp (hypocone). Figure 2 is a polarized transmitted light image of this cusp a t
higher magnification in which linear incremental markings in enamel can be seen
(above and left) parallel with the cusp margins in addition to three prominent lines
Fig. 1. Transmitted light micrograph of a n M’ buccolingual section illustrating the distal lingual cusp at left.
Field width = 2,730 pm.
Fig. 2. Transmitted light micrograph (Leitz 10)ofthe
distal lingual cusp of Figure 1 showing enamel and
dentine increments. Structural defects in dentine associated with the three vital labels (see Fig. 3 caption) are
indicated with white arrows and with minocycline administration in enamel by a black arrow. Field
height = 1,100 pm.
Fig. 3. Fluorescence image (Leitz 10) of Figure 2
illustrating vital labels in dentine (white arrows): rninocycline near to the enamel-dentine junction (originally yellow), DCAF near the pulp cavity (originally green),
and faint xylenol orange (originally orange) between
these two labels. Faint DCAF labeling (originally green)
of enamel could be seen circumferentially around cervical and cuspal enamel (black arrow: the optical density of
this green label is too near that of the surrounding field
in order to image this label rendered in a black and white
photomicrograph). Field height = 1,100 km.
coursing linearly within the dentine (white
arrows). Figure 3 illustrates the fluorescent
image of Figure 2, showing the labels in
dentine (minocycline closest to the enamel,
originally appearing yellow; DCAF furthest
from the enamel, originally appearing green;
xylenol orange, barely visible between these
two labels, originally appearing orange).
While the enamel originally exhibited a diffuse yellow-green fluorescence, one could
just see the corresponding DCAF label in
enamel running parallel with enamel increments close to the outer surface of the tooth
(Fig. 3, in the position of the black arrow).
While minocycline fluorescence was not seen
in enamel, it was evident a s a structural
defect over most of the molar cross section
(see Fig. 2., black arrow, and Discussion),
following precisely the course of this label in
dentine and arriving together with the dentine label a t the enamel-dentine junction in
several places along the crown section.
Figures 4 and 5 are higher magnification
transmitted light and fluorescent images,
respectively, of the same cusp. One can count
28 cross striations accounting for enamel
formed between the minocycline and DCAF
labels (Fig. 4, arrows), plus a n additional 1 8
cross striations from the time of the last label
to the time of death. The DCAF label was
visible a s a diffuse green band on the corresponding fluorescent image (Fig. 5, black
arrow; see also Fig. 3). Figure 6 is a graphic
representation of Figure 4 illustrating
enamel increments enumerated from the
enamel-dentine junction to the outer surface enamel. The average distance between
adjacent cross striations (the cross-striation
repeat interval) over the duration of cusp
development represented by the three labels
(hence 4 weeks) was 4.9 pm.
Specimen 2 did not present sufficient optical contrast for the purpose of enumerating
Fig. 4. Transmitted light micrograph (Neofluar 16)
of the cuspal region of Figure 2. The incremental structural defect associated with minocycline administration
is marked with a n arrow at bottom near the enameldentine junction. The subsequent 28th increment at the
time of administration of DCAF (see Fig. 5) is marked
with an arrow toward the cusp tip. Field height =
670 pm.
Fig. 5 . Fluorescence image (Neofluar 16)corresponding to the central area of Figure 4 illustrating the vital
labels in dentine (see Fig. 3). Faint DCAF labeling of
enamel was also observed a s in Figure 3 (black arrow)
and remains obscure in this black and white rendering.
Field height = 670 pm.
cross striations, thus disallowing detailed
examination of all enamel increments. However, the cross-striation repeat interval
within cervical enamel (compared with the
cuspal enamel measured in specimen 11,
which could be measured between enamel
increments meeting dentine labels a t the
enameklentine junction, provided a n average cross-striation repeat interval of 4.3 IJ-m.
This specimen was of further utility concerning dentine circadian periodicity. Figure
7 illustrates a transmitted light image of
buccal cervical dentine with structural defects (arrows; enamel is at bottom). Figure 8
is the corresponding fluorescent image with
the first DCAF label a t the bottom and the
others in sequence above. The arrow on Figure 8, which is in the same position of this
labels’ arrow in Figure 7, demarcates the
xylenol orange (third) label which is barely
visible. If one looks at, for instance, the
region between the minocycline (second) and
xylenol orange (third) labels between the
crosses of Figure 9, one can see 14 increments (von Ebner’s lines) between these two
labels with a n average 2.7 IJ-mbetween lines
(Fig. 9). Between all four labels and 6 weeks
of dentine formation between crosses of Figure 10,this repeat interval remains the same
(giving a total of 114 pm).
It was evident that some vital labels have
a systemic effect on the dentine and enamel
forming cells. The effect of minocycline administration was particularly evident a s a
structural defect in enamel (on these and
other specimens subsequently investigated).
For example, Figures 11and 12 are images of
a developing macaque molar cusp illustrating a n originally yellow minocycline label in
dentine (Fig. 11,arrow) and the corresponding structural defect in enamel (Fig. 12, arrow). Ideally one would hope to image the
vital labels employed here in the enamel
itself and, indeed, the fluorescent image did
reveal faint yellow minocycline labeling of
this structural defect. While DCAF administration rarely effected a marked structural
defect, it was often visible as a diffuse green
band in enamel (see Figs. 3 and 5).These are
the first reports of minocycline and DCAF
labeling of enamel.
While preliminary, the present study supports the earlier physicochemical documentation of enamel circadian periodicity by
Fig. 6 . Schematic of Figure 4 illustrating cuspal increments between minocycline and DCAF
labels (28 increments) and 18 subsequent increments of enamel formed toward the outer surface
enamel. No scale was used.
Mimura (1939) and Okada (1943). This corroboration was possible, in part, because of
altered dentine and enamel formation induced by the administration of fluorochromes. These systemic effects, appearing
as minor structural defects, occurred despite
the reported minimal interference of DCAF,
minocycline, and xylenol orange with bone
growth compared with other vital labels
(Newell-Morris and Sirianni, 1982).
Although demonstrations of vital labeling
in dentine are well known (e.g., Baker, 1972;
Kosugi, 1984; Suga, 19701,similar studies of
enamel are extremely rare. Baker (1972)
observed tetracycline labeling of enamel, not
as a discrete line as in dentine, but as a
diffuse band. Kosugi (1984) also observed
this phenomenon and confirmed the contigu-
ity of tetracycline labels in dentine with
those of the synchronously forming enamel.
Suga (1970) has suggested that tetracycline
labeling, once diffused within mineralizing
enamel, finally disappears because of the
vacuation of enamel organic matrix during
maturation mineralization. The coarse incremental lines observed in the present
study sample illustrates the systemic effect
that compounds such as minocycline have on
the cells’capacities to elaborate their organic
matrix. However, it is not clear whether the
cells participate in the deposition of the compounds.
Earlier studies of modern and fossil hominid enamel rhythms (e.g., Beynon, 1986;
Beynon and Dean, 1988; Beynon and Reid,
1987; Beynon and Wood, 1987; Bromage,
21 1
Fig. 7. Transmitted light micrograph (Neofluar 16)
of buccal cervical dentine of a n M' illustrating structural
defects associated with vital label administration (arrows; see Fig. 8). Enamel is at bottom. Field width =
670 pm.
Fig. 8. Fluorescence image (Neofluar 16)corresponding to Figure 7 illustrating vital labels in dentine: DCAF
at bottom and left nearest the enamel-dentine junction
(originally green), two subsequent minocycline labelsone adjacent to the DCAF label and one toward the pulp
cavity (originally yellowband a faint xylenol orange
label between these two minocycline labels (arrow: originally orange). The last minocycline label (nearest the
pulp cavity) was administered 4 days prior to sacrifice.
Field width = 670 pm.
1985,1987; Bromage and Dean, 1985; Dean,
1987a,b) assumed that enamel matrix
formed in daily increments, reflected in
enamel cross striations. The present study,
together with those of Mimura (1939) and
Okada (1943), confirms this assumption. At
the most fundamental level of investigation,
the enumeration of enamel circadian incre-
Fig. 9. Transmitted light micrograph (Neofluar 40)of cervical dentine increments between
two labels 14 days apart. Point to point measurement between these labels (crosses) equals 38
pm. Field width = 57 pm.
Fig. 10. Transmitted light micrograph (Neofluar 16)of cervical dentine increments between
four labels, each 14 days apart. Point to point measurement between the first and last labels
equals 114 pm.Field width = 143 pm.
ments represents a method for determining
the duration of life history periods over
which enamel is formed that depends not on
intra- or interspecific developmental varia-
tion. Thus the results of the present study
lend additional objective support to dental
developmental studies that employ enamel
incremental features in the data base.
I thank Laura Newell-Morris, who kindly
provided the study sample. This research
was supported (in part) by the L.S.B. Leakey
Trust, by grant number 668545 from The
PSC-CUNY Research Award Program of The
City University of New York, and by NIH
grants RR00166, DE02918, and RR05346 to
the Regional Primate Research Center a t the
University of Washington. The comments of
three reviewers and Alan Boyde’s enthusiasm to conduct this study (and within whose
laboratory this project got underway) are
gratefully acknowledged. I also thank Thomas Amorosi for the preparation of Figure 6 .
Fig. 11. Combined fluorescencefiinearly polarized
image (Leitz 10) of a minocycline labeled (arrow) and
subsequently alizarinated M, germ. Field height =
1,100 pm.
Fig. 12. Transmitted light micrograph (Leitz 10)of
Figure 11 illustrating the structural defect in enamel
relating to minocycline administration (arrow). One can
also see this defect within dentine in the position of this
label illustrated on Figure 11. Field height = 1,100 Fm.
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polychrome, macaque, increment, periodicity, pig, enamel, tissue, tailed, fluorescence, stud, dental, labeling, hard
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