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Microscopy of the neonatal line in developing human enamel.

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Microscopy of the Neonatal Line in Developing
Human Enamel
D E N N I S F. WEBER' A N D DALE R. E I S E N M A N N
Veterans Administration Hospital, Iowa City, Iowa and Department of
Oral Biology, T h e University of Iowa, College of Dentistry, Iowa City,
Iowa 52240 a n d University of Illinois, College of Dentistry,
Department of Histology, 808 S o u t h W o o d Street,
Chicago, Illinois
ABSTRACT
The neonatal line in developing human primary teeth was examined by means of phase contrast microscopy, microradiography and transmission electron microscopy. When thin sections were observed by light microscopy,
the lateral dimensions of the line were not as extensive as had been previously
reported. The line had a "staircase" configuration and appeared to be identical to
published light micrographs of the stria of Retzius. On radiograms, the lateral
extent of the hypomineralization was also decreased. The ultrastructural basis for
the neonatal line appeared to be a localized change in configuration of enamel
prisms along with a possible reduction in crystal concentration. The possibility
that some rods actually end at the line could not be excluded, however.
A number of the classical morphological
features of enamel seen with the light microscope have not been adequately defined
on an ultrastructural basis. Among these
are the incremental lines of Retzius which
still bear the name of the man who first
described them (Retzius, 1837). Although
several ultrastructural descriptions of the
Retzius lines are present in the literature,
they must be considered tentative for at
least two reasons: they were either based
on observations of partially decalcified material (Hinrichsen and Engle, '66) or the
electron microscopic findings were not correlated with light microscopic results
(Frank, '65; Frank and Nalbandian, '67).
Neonatal lines are prominent incremental
lines which are formed shortly after birth
(Rushton, '33; Schour, '36). One neonatal
line is usually present in all human primary teeth and occasionally one is found
in the first molars of the secondary dentition. The Gustafsons ('67) have classified
the neonatol line as a type of pathological
Retzius line, i.e., a Retzius line produced
by some alteration other than the bending
of the rods. Attempts to determine the
structural nature of the neonatal line using
various light microscopic methods have
produced conflicting results. Rushton ('33)
suggested that there was either a hyper- or
hypomineralization of the interprismatic
AM. J.
ANAT.,
132:
375-392.
substance within the line. In a later study,
Rushton ('39) stated that the portions of
the prisms within the line were more highly
mineralized than the portions of the same
prisms on either side of the line. Sognnaes
('49) described the neonatal line as being
comprised of thickenings of the prism
sheaths. Gustafson ('59) described an increase in the amount of interprismatic
substance within the line with the consequent narrowing of that portion of the
prism passing through the line. Radiographically, the neonatal line has been
reported to be relatively radiolucent and
therefore hypomineralized (Crabb, '59;
Allan, '60, '67; Silness, '69). No information is presently available concerning the
ultrastructure of the neonatal line region.
Since the neonatal line is a very prominent incremental line and the enamel of
the primary teeth is relatively free of other
incremental lines, we felt that this isolated
situation presented an ideal set of conditions for a correlated light and electron
microscopic study of a particular type of
Retzius line.
MATERIALS AND METHODS
Twenty-five developing human primary
teeth (10 incisors, 6 canines and 9 molars)
1 Forward reprint requests to: Dr. Dennis F. Weber,
Veterans Administration Hospital, Building 3, Room
224, Iowa City, Iowa 52240.
375
376
DENNIS F. WEBER AND DALE R. EISENMANN
were utilized. The teeth were obtained from
two infants: a 60-day old male and a 75day old female. The male was delivered
after a normal gestation period and the
female was delivered prematurely at six
and one-half months. The interim medical
histories of both infants were essentially
unremarkable. The gross autopsy findings
in both infants were typical of the so-called
“crib-deaths” of infancy. A number of other
teeth from infants who had lived for varying periods of time up to two and one-half
days as well as teeth from a n individual
who died at birth after a gestation period
of ten months were also studied. After
careful removal from their bony crypts, all
of the teeth were fixed in absolute alcohol.
Light microscopy. The teeth were embedded in methyl methacrylate using the
method described by Yaeger (‘58). Initially.
the embedded teeth were sectioned longitudinally in a labiolingual plane a t 150
using a thin sectioning machine which
employed a revolving diamond edge wheel.
Usually, the sections were taken from central regions o f the crowns where the enamel rods run parallel to this plane of sertioning. Thirty sections were mounted,
without further reduction, on microscope
slides in a synthetic mounting medium for
viewing with ordinary transmitted light.
Three other thick sections were radiographed using Siemen’s microradiographic
unit equipped with a copper target and a
nickel filter and operated at 20 KV.
Twenty-five of the thick sections were
using a reduction
reduced to 4 to 25
technique described by Fremlin et al. (’61 ).
These sections were mounted in either immersion oil or a synthetic mounting medium and observed by phase contrast
microscopy. Twenty thick sections were
reduced ( 5 to 25 p ) for microradiography
using a n integrated mounting and reduction technique (Weber, ’67). The microradiograms of the thin sections were obtained with a unit which was custom built
to the specifications of the discontinued
Philips CMR unit. A Philips tube equipped
with a tungsten anode and a beryllium window was used. Exposures were made a t
5 KV and 2 MA. The microradiograms were
mounted on microscope slides in a synthetic mounting medium.
Electron microscopy. Seven teeth were
embedded either in methyl methacrylate or
Araldite. After polymerization the block
was trimmed to include the line area. A
saturated solution of dibasic calcium phosphate was used in the sectioning trough to
prevent decalcification (Boothroyd, ’64).
Sections were stained with uranyl magnesium acetate and examined in a Hitachi
7 s electron microscope.
OBSERVATIONS
Light microscopy. When thick sections
were viewed by transmitted light with the
objective focused on the upper surface of
the section, the appearance and lateral dimensions of the line varied according to
the angle a t which the rods were sectioned
and the inclination of the line relative to
the plane of sectioning. Under low magnification, in regions where the rods were cut
longitudinally, the line appeared to be a
dark linear band running obliquely across
the surface of the enamel (fig. 1). I n
regions where the rods were cut obliquely
(e.g., gnarled enamel in the cusp tips) and
the inclination of the line relative to the
plane of sectioning was less than go”, the
line had a more diffuse appearance as well
as apparently greater lateral dimensions
(fig. 1). Under higher magnification, in
regions where the rods were sectioned
longitudinally, the line again appeared as
a broad (20 to 30 p ) dark band. Its periphery had a scalloped or serrated appearance
(fig. 2 ) . Continuity of the rods seemed to
be temporarily interrupted at the line.
Changes i n orientation of the rods at the
line were not apparent. Modifications of
the morphology of the prisms or prism
boundaries within the line were difficult to
assess because of the thickness of the
sections.
When thinner sections (20 p ) were
viewed by phase contrast microscopy, the
appearance of the line changed dramatically. The broad lateral dimensions were
no longer present. The line had a “staircase’’ configuration which appeared to be
built up of a series of prominent dark cross
striations (figs. 3 , 4). Extremely thin sections ( 4 p ) viewed under higher magnification further clarified the “staircase” appearance (fig. 5 ) . The staircase was made up
of dark cross striations and segments of
MORPHOLOGY OF THE NEONATAL LINE
the prism boundaries, both of which were
approximately 1 in width.
Microradiograp hy . In micror adiograms
of the thick sections the line appeared as
a broad (20 to 40 p ) radiolucent band
whose peripheral margins were relatively
diffuse (fig. 6). Microradiograms of successively thinner sections showed a decrease in the width and prominence of the
line (figs. 7, 8). In addition, the line did
not appear to be uniformly radiolucent as
many radiopaque line patterns traversed
the neonatal line. In microradiograms of
the thinnest sections (fig. 8), the line appeared to be composed of accentuations of
the radiolucent prism boundaries which
traversed the entire width of the enamel.
Under optimum conditions, which weie
difficult to reproduce photographically,
there also appeared to be other radiolucent
regions within the line. They corresponded
in position to the cross striations seen with
phase microscopy. Some of these are
faintly visible in figure 8.
Phase microscopic and microradiographic observations of teeth taken from
the infants who had survived for periods
up to two and one-half days revealed no
evidence of the formation of a neonatal
line. No neonatal lines were seen in the
teeth taken from the ten-month in utero
specimen.
Electron microscopy. The most surprising ultrastructural feature of the neonatal
line was its inconspicuousness. In many
specimens the line could hardly be seen
with the electron microscope even though
it could be visualized clearly with the dissecting microscope on the surface of the
block of enamel from which the sections
were taken. Ultrastructurally, the line appeared to consist of a thin crystal-deficient
region running in a tortuous course
obliquely across the enamel prism (figs. 9,
10). The line was continuous with abutting prism sheaths and similar to them except for a slightly greater thickness than
the typical prism sheath. The crystaldeficient region was not always continuous
across the prisms. Some prisms appeared
to traverse the line region. Such prisms
appeared to undergo a dramatic change in
configuration (figs. 10, 11).
Figures 9 and 10 illustrate a neonatal
line in which enamel along either side of
377
the line contains areas with abnormal crystal patterns. This was observed inconsistently in several specimens. When present,
the abnormal crystal patterns existed as
scattered regions of decreased crystal concentration in proximity to the neonatal
line.
High magnification of the line revealed
a fine granular material occupying the
space between the crystalline borders of
the pre- and postnatal enamel (inset of
fig. 10). No apparent differences in the
morphology of crystals of pre- and postnatal enamel were observed.
DISCUSSION
It is possible that unusual metabolic disturbances or trauma occurring at birth or
during the neonatal period might influence
the structure of the neonatal line. Since
the teeth studied in the present investigation were taken from infants who had normal histories during these periods, we assume that the neonatal lines studied in the
present investigation were representative.
The presence of a neonatal line in the
75-day old individual who was born after
a six-and-one-half-month gestation period
and the absence of the line in the teeth
from the individual who died at birth after
a ten-month gestation period further confirms the fact that the formation of the line
is associated with a particular event (birth)
rather than a period of time or stage of
development. On a light microscopic level,
no indication of the formation of a neonatal line was seen in any of the individuals who had lived for up to two and onehalf days. Though its general association
with birth is certainly well established, the
exact time at which the line is formed or
period during which it is forming is not
known and, indeed, may vary.
There was little correlation between our
observations of thin ground sections of
enamel using phase contrast microscopy
and microradiography and previous light
microscopic descriptions of the neonatal
line. No thickening of the prism boundaries
or prism sheaths or gross bending of the
rods was evident. There was, however,
some evidence of constriction of the portins of the prisms within the line (fig. 5).
However, the constriction of a rod was not
compensated for by an expansion of adja-
378
DENNIS F. WEBER AND DALE R. EISENMANN
cent prism boundaries but rather by an “prism boundary” light microscopically.
expansion of the adjacent rod. Also, pre- Ultrastructural examination of the prism
vious radiographic images of the line have sheaths that pass through the neonatal line
shown a gross distortion of the lateral ex- (figs. 9, 10) indicates only minor increases
tent of hypomineralization. The staircase in thickness but substantial changes in
appearance of the line corresponded with direction of the sheaths.
previous descriptions of Retzius lines.
The granulatr material observed within
Gwinnet (’66) and Poole (’67) have shown the neonatal line lends support to the conlight photomicrographs of thin sections of tention that a crystal-deficient region acmature enamel in which the “stepped’ ap- tually exists and is not a shrinkage artifact.
pearance of the incremental line was eviAlthough the apparent degree of hypodent. Gwinnet reported that this configura- mineralization of the neonatal line detion did not represent the typical Retzius creases markedly with section thickness,
line but rather that it occurred in only there is still evidence of a diffuse hypomin15% of the sections which he examined. eralization approximately 10 wide in the
Therefore i t is suggested that the light mi- thinnest sections examined (fig. 8). Eleccroscopic and ultrastructural characteristics of the neonatal line described in the tron dersity of enamel cannot be fully
present paper may also apply to other relied upon as an indicator of mineral denRetzius lines. This idea is supported by a sity. However, the observation of a dedescription given by Frank and Nalbandian creased concentration of crystals along
(’67) : “Concerning the incremental lines either side of some neonatal lines may
of Retzius, we encountered on a longitudi- provide an explanation for the diffuse
nal section through human foetal enamel hypominer alization.
Previous light microscopic studies rea band of uncalcified organic matrix 20004000 A thick, running across the rods. It vealed changes in enamel prism configurawas continuous through the rod and inter- tion within the neonatal line. Because of
rod substance and assumed an ultrastruc- the highly variable outline of prisms which
ture similar to that of the rod sheath.” This became fully apparent only at the ultrais not at all unlike our ultrastructural de- structural level, it was difficult to describe
scription of the neonatal line in the fetal thoroughly the nature of changes in their
enamel.
gross morphology at the line as compared to
The ultrastructural appearance of elsewhere in enamel. Electron mizroscopy
enamel conforms with earlier electron mi- has revealed a profound change in configcroscopic descriptions of this tissue. Prism uration of prisms at the neonatal line.
sheaths are approximately 1000 A wide as Constrictions or expansions of the prisms
described previously (Boyde, ’64). The are largely compensated for by compleprism boundary as described light micro- mentary changes in adjacent prisms. This
scopically is much thicker (0.5-1 p ) and localized alteration in form which involves
is therefore not considered a true repre- the respective sheaths may also be at least
sentation of the sheath (Helmcke, ’67). It partially responsible for what appears to
has been demonstrated that this discrep- be a slight thickening of the sheaths, in
ancy is caused by a summation effect that it is simply cut at more oblique angles
which occurs when a curved structure such here and appears thickened.
as the sheath is examined in sections which
The ultrastructural basis for the neoare much thicker than the sheath itself. natal line may be no more complex than a
The curvature and lateral deviations of the localized change in configuration of
sheath within a section lead to the produc- enamel prisms along with a possible retion of a widened summation image by duction in the concentration of crystals.
light or x-rays as they pass through the We cannot exclude, however, the possisection (Glas and Nylen, ’65). Any in- bility that some rods actually end at the
crease in the waviness of the sheath, such neonatal line. Light microscopic and microas occurs in the area of the neonatal line radiographic descriptions of characteristics
in figures 9 and 10, will appear as an in- such as marked thickening of prism boundcreased width or prominence of the aries and excessive lateral extension of
MORPHOLOGY OF THE NEONATAL LINE
the line are apparently products of section
thickness and optical aberrations.
LITERATURE CITED
Allan, J. H. 1959 Investigations into the mineralization pattern of human dental enamel.
J. Dent. Res., 38: 1096-1128.
1967 Maturation of Enamel. In: Structural and Chemical Organization of Teeth. Vol.
1. A. E. W. Miles, ed. Academic Press, New
York, pp. 467-494.
Boothrovd. B. 1964 The moblem of demineralization i n thin sections of fully calcified bone.
J. Cell. Biol., 20: 165-173.
Boyde, A. 1964 The structure and development
of mammalian enamel. Ph.D. Thesis, University of London.
Crabb, H. S. M. 1959 The pattern of mineralization of human dental enamel. Proc. R. SOC.
Med., 52: 118-122.
Engstrom, A., R. C. Greulich, B. L. Henke and B.
Lundberg 1957 High-resolution contact microradiography with ultra-soft polychromatic
X-rays. Xn: X-ray Microscopy and Microradiography. V. E. Cosslett and H. H. Patee, eds.
Academic Press, New York, pp. 218-233.
Frank, R. M., and J. Nalbandian 1967 Ultrastructure of amelogenesis. In: Structural and
Chemical Organization of Teeth. Vol. 1. A. E. W.
Miles, ed. Academic Press, New York, pp.
399-466.
Fremlin, J. H., J. Mathieson and J. Hardwick
1961 The preparation of thin sections of dental enamel. Arch. Oral Biol., 10: 55-60.
Glas, J. E., and M. U. Nylen 1965 A correlated
electron microscopic and microradiographic
study of human enamel. Arch. Oral Biol.,
10: 893-908.
Gustafson, A. G. 1959 A morphologic investigation of certain variations in the structure and
mineralization of human dental enamel. Odont.
Tidskr., 67: 361-472.
Gustafson, G., and A. G. Gustafson 1967 Microanatomy and histochemistry of enamel, In:
~
379
Structural and Chemical Organization of Teeth.
Vol. 11. A. E. W. Miles, ed. Academic Press,
New York, pp. 135-162.
Gwinnett, A. J. 1966 Histology of normal
enamel. 111. Phase contrast study. J. Dent.
Res., 45: 865-869.
Helnncke, J. G. 1967 Ultrastructure of enamel.
I n : Structural and Chemical Organization of
Teeth. Vol. 11. A. E. W. Miles, ed. Academic
Press, New York, pp. 135-162.
Hinrichsen, C. F. L., and M. B. Engel 1966 Fine
structure of partially demineralized enamel.
Arch. Oral Biol., 11: 65-93.
Poole, D. F. G. 1967 Physiology of tooth tissues: enameloid and enamel in recent vertebrates, with a note o n the history of cementum.
In: Structural and Chemical Organization of
Teeth. Vol. 1. A. E. W. Miles, ed. Academic
Press, New York, pp. 111-147.
Retzius, A. 1897 Microscopic investigation of
the structure of the teeth. Arch. Anat. Physiol.,
8: 486-519.
Rushton, A. 1933 On the fine contour lines of
the enamel of milk teeth. Dent. Rec., 53:
170-1 71.
1939 The birefringence of deciduous
tooth enamel formed before and after birth.
Brit. Dent. J., 67: 1-10.
Schour, I. 1936 The neonatal line i n the
enamel and dentin of human deciduous teeth
and first permanent molar. J. Am Dent. Assoc.,
23: 1946-1955.
Silness, J. 1969 Some variations in the microradiographic appearance of human deciduous
enamel. Odont. Rev., 20: 93-109.
Sognnaes, R. F. 1949 The organic elements of
the enamel. 111. The pattern of the organic
framework in the region of the neonatal and
other incremental lines of the enamel. J. Dent.
Res., 28: 558-564.
Weber, D. F. 1967 Microradiography of thin
sections of mineralized tissue. Arch. Oral Biol.,
12: 355-358.
PLATE 1
EXPLANATION O F FIGURES
Two light photomicrographs of thick sections taken with the aperature
diaphragm partially closed and the objective focused on the upper surface
of the section. D, dentin; E, enamel.
Low power view of a portion of the mesiobuccal cusp of a lower first
primary molar. The nconatal line can be seen running obliquely across
the surface of the enamel. In regions where the rods were cut longitudinally (opposing arrows), the neonatal line appeared as a dark
linear band. In regions where the rods were cut obliquely (single
arrow), the line had a more diffuse appearance as well as apparently
greater lateral dimensions. x 175.
Higher power view of the region indicated by the opposing arrows in
figure 1. The neonatal line (between opposing arrows) appears as a
broad band 20-30
wide. The rods in the prenatal enamel ( p ) appear
to terminate a t the line only to resume their course again i n the postnatal enamel (p’). All sections studied by other methods were taken
from an aiea similar to this. X 350.
380
MORPHOLOGY OF THE NEONATAL LINE
Dennis F. Weber and Dale R. Eisenmann
PLATE 1
PLATE 2
EXPLANATION O F FIGURES
Three phase contrast photomicrographs of thin ground sections.
382
3
Low power view of a section 20 g thick. The neonatal line is located
between the opposing arrows. Note the “staircase” appearance of the
line and the decrease in lateral dimensions of the 1i:ne when compared
to that seen in figure 2. X 250.
4
High power view of the same section taken from the region indicated
by the opposing arrows. The horizontal arrow indicates a dark cross
striation and the vertical arrow indicates a bright prism boundary.
x 1000.
5
High power view of a n extremely thin ground section ( 4 p). The
“staircase” appears to be made up of the dark cross striations (horizontal arrow) and segments of the bright prism boundaries (vertical
arrow). x 2500.
MORPHOLOGY OF THE NEONATAL LINE
Dennis F. Weber and Dale R. Eisenmann
PLATE 2
383
PLATE 3
EXPLANATION O F FIGURES
Three niicroradiograins of successively thinner ground sections.
Microradiogram of a thick section. The neonatal line appears as a
broad radiolucent band. The inner and outer limits of the line are
indicated by the opposing arrows. The peripheral margins of the line
are relatively diffuse. x 250.
Microradiogram of a thinner section ( 2 5 r ) . The neonatal line is located between the opposing arrows. Note the reduction in the width
of the hypomineralized neonatal line. x 250.
Microradiogram of a n extremely thin section ( 4 f i ) . The neonatal line
region is located between the opposing arrows. Small arrow indicates
a radiolucent cross striation seen with phase contrast microscopy.
X 250.
384
MORPHOLOGY OF THE NEONATAL LINE
Dennis F. Weber and Dale R. Eisenmann
PLATE 3
385
PLATE 4
EXPLANATION OF FICURE
The electron micrograph is mounted with the prenatal enamel i n the
upper part of the illustrations and the postnatal enamel in the lower part.
9
386
Electron micrograph of the neonatal line region. The crystal-deficient
line runs horizontally acioss the micrograph (arrow). Note the region
of lower concentration of crystals along either side of the wavy neonatal line. x 5500.
MORPHOLOGY OF THE NEONATAL LINE
Dennis F. Weber and Dale R. Eisenmann
PLATE 4
387
PLATE 5
EXPLANATION OF FIGURE
The electron micrograph is mounted with the prenatal enamel in the
upper part of the illustrations and the postnatal enamel in the lower part.
10
388
High magnification of right-hand central portion of figure 9. Note
what appears to be the abrupt termination of the prism which meets
the neonatal line i n the center of this illustration. Arrow 1, prism
sheath separating adjacent prisms; arrow 2, region of neonatal line
in inset x 9900. Inset: high magnification of a small segment of the
line showing a granular material within the crystal-deficient region.
x 44,000.
MORPHOLOGY OF THE NEONATAL LINE
Dennis F. Weber and Dale R. Eisenmann
PLATE 5
389
PLATE 6
EXPLANATION O F FIGURE
The electron micrograph is mounted with the prenaltal enamel in the
upper part of the illustrations and the postnatal enamel i n the lower part.
11
390
The neonatal line can be seen following a tortuous path horizontally
across the lower one-third of the micrograph. This micrograph illusstrates that some prisms (prism a ) pass through the line region. When
doing so they appear to undergo a severe change in configuration. Note
the constriction or translocation of prism a i n the region indicated by
the arrow. Other prisms i n the field (prisms b and c ) appear to
terminate at the crystal-deficient region. x 6700.
MORPHOLOGY OF T H E NEONATAL LINE
Dennis F. Weber a n d D a l e R. E i s e n m a n n
PLATE 6
391
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