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The presence of fenestrated capillaries in the papillary layer of the enamel organ.

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The Presence of Fenestrated Capillaries in the
Papillary Layer of the Enamel Organ
Harvard School of Dental Medicine, Boston, Massachusetts
The capillary network of the papillary layer of the enamel organ was
studied with the electron microscope. Mandibular incisor teeth of ten-day old Swiss
albino mice were fixed in formaldehyde-glutaraldehyde, postfixed in osmium tetroxide
and prestained with 0.5% uranyl acetate prior to being embedded in Epon. Examination of ultrathin sections revealed the presence of fenestrations approximately 500 A
to 700A in diameter within the most attenuated regions of the capillary walls. In
most instances the fenestrations appeared to be closed by diaphragms usually consisting of a thin membrane with a thicker centrally located button of electron opaque
The presence of fenestrated capillaries within the papillary region of the enamel
organ lends further support to the concept that papillary cells are engaged in transport
functions during the maturation of enamel.
The development and intimate apposition of a capillary network to the ameloblastic layer of developing teeth has been
the subject of numerous histological studies in a wide variety of species (Addison
and Appleton, '22; Bernick, '60; Glasstone,
'62; Jordan, '21; Jump, '38; Kingery, '24).
In rodent incisor teeth an especially abundant network of capillaries becomes closely
apposed to the enamel organ overlying the
post-secretory ameloblasts (Adams, '62).
This region of the enamel organ, commonly referred to as the papillary layer,
has an architecture of alternating epithelial
ridges and capillaries which provides an
increased surface area for the rapid exchange of metabolites (Reith, '59; Kallenbach, '66).
In the only reported electron microscopic
study of the vascular supply to the enamel
organ, Decker ('67) has described unfenestrated capillaries of the conventional
or continuous type in close proximity to
the external enamel epithelium of the developing rat molar. The present investigation, however, revealed that the capillaries
associated with the papillary layer of the
mouse incisor are of the fenestrated variety. Fenestrated capillaries have been reported within numerous organs, many of
which are specialized for the rapid exchange of metabolites. A few examples
are the kidney (Pease, '55; Yamada, '55;
Rhodin, '62), the lamina propria of intesANAT. REC., 1 6 3 ; 71-80.
tinal villi (Palay and Karlin, '59; Bennett,
Luft and Hampton, '59), parotid gland
(Parks, '61), and the rete mirabile of fish
swim bladder (Fawcett, '63). For a
thorough compilation of the reports of
fenestrated capillaries one should consult
a review of the ultrastructure of the vascular membrane by Majno ('65).
Mandibular incisors of ten-day old Swiss
albino mice were dissected free of surrounding tissues in a dish containing a formaldehyde-glutaraldehyde fixative buffered to pH
7.4 with cacodylate (Karnovsky, '65). Upon
completion of the dissection, the incisors
were cut into smaller pieces and transferred to a fresh solution of fixative. After
30 minutes in this fresh solution at room
temperature, the tissues were transferred
through several rinses of 0.1 M cacodylate
buffer and stored overnight in buffer containing 0.25 M of sucrose. Postfixation
was carried out in a 1% collidine buffered
osmium tetroxide solution (pH 7.4) for
45 minutes at 4°C. The tissue blocks were
then stained at room temperature with
0.5% uranyl acetate in acetate-Verona1
buffer for 45 minutes (Farquhar and
Palade, '65). Dehydration was rapidly
carried out in a graded series of ethanol
and the tissues subsequently embedded in
Epon (Luft, '61). Sections were cut on
Received July 19, '68. Accepted Sept. 25, '68.
a Porter-Blum microtome equipped with a
diamond knife, stained with lead citrate
and examined in an RCA-EMU-3G electron microscope at 50 kv.
The portions of the enamel organ overlying post-secretory ameloblasts, when
viewed in sections cut perpendicular to the
long axis of the incisor, are arranged into
a series of papillae projecting away from
the ameloblastic layer (fig. 1 ). Capillaries
originating in the connective tissue of the
surrounding dental sac course within the
spaces between adjacent papillae (fig. 1).
Tangential sections through the papillary
layer reveal the complex network of capillaries in close association with the papillary cells of the enamel organ (fig. 2).
With the exception of the nucleus and
perinuclear cytoplasm, which usually
bulged outward into the lumen of the
capillary, the cytoplasm of the endothelial
cell was extended to form the narrow attenuated capillary wall (fig. 3). The narrowest
portions of the endothelial wall (300 A
to 500 A thick) were frequently interrupted
by fenestrations approximately 500 A to
7 0 0 A in diameter (figs. 4,5,6). The majority of the fenestrations were closed by
diaphragms (figs. 4, 5, 6). In many instances the diaphragms were characterized
by a centrally located button of electron
opaque material surrounded by a tenuous
30 to 4 0 A membrane (figs. 8, 9). This
arrangement was verified by sections cut
tangential to a fenestration (fig. 10). On
occasion similar buttons of electron opaque
material could be detected at the mouths
of forming pinocytotic vesicles (figs. 8, 9).
A basement membrane, approximately
300 to 500A thick, was present adjacent
to the capillary walls and adjacent to papillary cells of the enamel organ (fig. 3).
Small collagen fibrils and material of the
same structure and electron opacity as
that which was observed to constitute the
basement membrane were usually present
within the narrow extracellular space separating the vasculature from the enamel
organ (figs. 3, 8, 9).
As described in greater detail in a previous publication (Garant and Nalbandian,
'68) the papillary cells abutting the capillaries were usually characterized by num-
erous cytoplasmic infoldings and microvilli which serve to increase the surface
area, and, in addition, they contained
numerous well developed mitochondria
and coated vesicles, suggesting a possible
transport function.
Because the papillary cells are strategically placed between the ameloblastic layer
and the rich plexus of capillaries located
within the dental sac, it is not surprising
that numerous investigators have hypothesized that they were in some way involved
in the movement and/or concentration of
nutrients needed during enamel formation
(Adams, '62; Addison and Appleton, '22;
Bernick, '60; Marsland, '52; Reith and
Cotty, '62; Williams, '23).
Electron microscopic studies of the papillary layer in both the rat and mouse have
shown that the cells making up this tissue
contain large numbers of mitochondria,
numerous coated vesicles and a rather extensive surface area due to the development of many microvilli and surface plications, all of which suggest a specialization
for transport functions (Elwood and Bernstein, '65; Kallenbach, '66; Garant and
Nalbandian, '68). The presence of a rich
network of fenestrated capillaries in close
association with the cells of the papillary
layer provides additional evidence that this
tissue performs a transport function during amelogenesis.
During the calcification or maturation
phase of enamel formation there is a reported increase in the mineral content
and a decrease in the water and organic
component of the enamel matrix (Weinmann, Wessinger, and Reed, '42; Deakins,
'42)- Electron microscopic studies of postsecretory ameloblasts have suggested that
they are involved in the removal of organic
matrix during enamel maturation (Reith,
'61, '63), and more recently autoradiographic investigations have provided evidence that this component of the matrix
may be a sulfated compound (Reith and
Cotty, '67). Perhaps the cells of the papillary layer are engaged in functions which
lead to the increased mineral and decreased water content found within mature enamel as proposed by Adams ('62).
The preferential distribution of fenestrated capillaries to certain organs known
to be involved in the modification of body
fluids suggests that they are structurally
specialized for rapid exchange of fluid and
solutes (Majno, ’65). Glomerular capillaries are known to pass fluid 100 times
more rapidly than the unfenestrated capillaries of striated muscle (Pappenheimer,
Renkin, and Borrero, ’51). It has been
proposed that the fenestrations and the
intercellular clefts represent the morphological equivalent of the physiologist’s
small pore, and that the higher permeability measured in fenestrated capillary beds
is due to the larger and thinner surface
area available for exchange provided by
the diaphragmatic membranes of the fenestrations (Luft, ’65). The manner in which
fenestrations are formed and the nature
and origin of the diaphragms are speculative. Luft (’65) has proposed that the
fenestrations arise by fusion of a cytoplasmic vesicle with the endothelial cell membrane, followed by degeneration of the
inner and middle layers of the membrane,
while the outer layer remains intact as a
thin diaphragm stretched across the mouth
of the vesicle. Contact of the vesicle with
the other surface of the endothelial wall
and rupture of all three layers of the membrane at this site completes the fenestration. The observations of “buttons” or
diaphragms at the mouth of vesicles (fig.
9) and structures interpreted as vesicles
with double diaphragms (fig. 8) in this
study may be interpreted as stages in the
formation of fenestrations according to the
scheme proposed by Luft (’65).
Although the fenestrations observed
within the capillaries of the papillary layer
of the mouse enamel organ are fewer in
number per available surface area than
those reported within renal capillaries
(Rhodin, ’62), it can be assumed that they
provide for more rapid exchange than continuous or unfenestrated capillaries. Thus
the papillary layer is closely apposed to a
capillary network containing the morphological characteristics usually equated with
one specialized for increased permeability.
This evidence further substantiates the
current concept that the extra-ameloblastic
portions of the enamel organ are engaged
in some transport activity during the maturation of enamel.
The authors wish to express their thanks
to Dr. John Nalbandian for his critical
evaluation of the manuscript and to Mr.
Allen Peeler for his technical assistance.
This work was supported in part by
U.S.P.H.S. research grant DE 01766 and by
training grant DE 00111 from the National Institute of Dental Research, National Institutes of Health.
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Light micrographs of the papillary layer of the mouse incisor cut perpendicular (fig. 1) and parallel (fig. 2) to the developing enamel surface.
1 Alternating papillary projections of epithelial cells ( P ) and capillaries
( C ) overlying the reduced (post-secretory) ameloblasts (A). DS, dental sac; E, enamel. x 720.
2 Close apposition of the papillary cells (P) with the rich network of
capillaries (C). X 730.
Philias R. Garant and Roberta Gillespie
Electron micrographs of a portion of a fenestrated capillary in the
papillary region of the enamel organ.
Low power electron micrograph illustrating the attenuated endothelial wall ( E ) and portions of the closely apposed papillary cells (P).
Outlined rectangular areas are presented at higher magnifications
in figures 4, 5, and 6. BM, basement membranes; L, lumen; hl,
mitochondria; MV, microvilli; RBC, red blood cell. x 15,600.
High power electron micrographs of the endothelial wall containing
randomly spaced fenestrations (arrows). Note the presence of thin
diaphragms stretched across the fenestrations. Figures 4 and 5,
x 60,000. Figure 6, X 63,000.
Philias R. Garant and Roberta Gillespie
High power electron micrographs of the fenestrated endothelial wall.
The continuity of the capillary wall is interrupted by numerous
fenestrations (arrows). Note the many microvilli ( M V ) which increase the surface area of the adjacent papillary cell. x 40,000.
8 Portion of the endothelial wall containing two fenestrations (arrows)
one of which appears to be closed by a double diaphragm. BM,
basement membrane; Col, collagen fibers; L, lumen. X 45,000.
Two fenestrations (arrows) each with a centrally located button of
electron opaque material. Vesicle ( V ) possessing a similar diaphragm
and button ( B ) across its mouth. BM, basment membrane material;
L, lumen. x 114,000.
10 Fenestrations cut a t different angles illustrating their circular structure and centrally located button. CS, cross section; OS, oblique section; TS, tangential section. X 101,000.
Philias R. Garant and Roberta Gillespie
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presence, capillaries, papillary, layer, enamel, organy, fenestrated
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