/ . Embryo!, exp. Morph. Vol. 48, pp. 93-100, 1978
Printed in Great Britain © Company of Biologists Limited 1978
93
The mechanical role of the cranial base in palatal
shelf movement: an experimental re-examination
By L. L. BRINKLEY 1 AND M. M. VICKERMAN
From the Department of Anatomy, Medical School and the Department
of Oral Biology, School of Dentistry, The University of Michigan
SUMMARY
Straightening of a cranial base flexure observed in the midsagittal presphenoid region of
mice has been postulated to play a major role in palate closure by providing the internal shelf
force for palatal shelf movement. The straightening is thought to take place during the 8 h
immediately preceding closure. This mechanical model assumes that straightening of the
cranial base flexure is transmitted laterally to the alar regions of the sphenoid and then
downward to the palatal shelves. According to this hypothesis, if theflexuredid not straighten
or the force generated by this form change was not transmitted to the palatal shelves,
movement to the horizontal plane should not take place. Physical disruption of the area of
the flexure should remove the potential for form change through force transmission. SwissWebster mouse fetuses which were 18 or 24 h prior to palate closure were obtained and the
heads removed. Tongue and brain were then removed from each head. In the experimental
specimens a midsagittal lesion, averaging 407 /tm in length and 84 /tm in width, extending
from the nasal septum through the craniopharyngeal area was made. This lesion ablated the
flexure region of the cranial base far in advance of the time straightening is thought to take
place. Heads of litter-mates with intact cranial bases served as controls. Specimens with
intact and ablated flexures were suspended in a submerged, circulating culture system
which was continuously gassed. After 18 h of culture, palatal shelf elevation had occurred
in fetuses of both ages whether or not their cranial bases were intact. Palatal shelf elevation in vitro does not require an intact cranial base.
INTRODUCTION
The cranial base provides the skeletal superstructure to which are attached
many orofacial structures, including the palatal shelves. Several investigators
have noted a cranial base flexure in the presphenoid region prior to closure,
which has straightened after closure occurs (Harris, 1964,1967; Verrusio, 1970;
Larsson, 1972; Hart, Smiley & Dixon, 1972; Taylor & Harris, 1973; Long,
Larsson & Lohmander, 1973). The initial observations of Harris (1964) led
Verrusio to postulate t h a t ' . . . straightening of the cranial base plays a role in
palate closure by providing the "internal shelf force"' (Verrusio, 1970).
Specifically, the mechanical model assumes that changes in the shape of the
1
Author's address: Department of Anatomy, Medical School, The University of Michigan
Ann Arbor, Michigan 48109. U.S.A.
7
EMB 48
94
L. L. BRINKLEY AND M. M. VICKERMAN
cranial base are transmitted to the passive palatal shelves, causing them to
assume a horizontal position. This hypothesis implies that any activities occurring in the midline flexure area of the cranial base which could result in a mechanical force, must be transmitted laterally to the alar regions of the sphenoid,
then downward to the attached palatal shelves. A corollary of this hypothesis
is that the cranial base must be intact for the shape change to have any effect on
the attached palatal shelves.
Although several descriptive studies have reported findings on the mechanical
role of the cranial base prior to or during palate closure, no direct experimental
manipulations of the cranial base have been undertaken. The recent design and
testing of a unique in vitro technique which allows palate closure in fetal mouse
heads with the tongue and brain removed (Brinkley, Basehoar, Branch &
Avery, 1975) now permits direct experimental alteration of the cranial base.
Using this system, the present study was performed to determine if disruption
of the physical continuity of the cranial base would affect palatal shelf movement.
MATERIALS AND METHODS
Animals. Thirteen-day pregnant Swiss-Webster mice (plug day = 0) were
obtained from Spartan Research (E. Lansing, Michigan). Two ages of fetuses
were used, 24 h and 18 h prior to palate closure based upon morphological
criteria (Walker & Crain, 1960, Brinkley et al. 1975). Some variation in the
developmental stage of litter-mates was found, thus to ensure the greatest
possible uniformity those markedly advanced or retarded from the norm were
discarded.
The mice were killed at the selected times by cervical dislocation, and all
subsequent operations were carried out in an Edgegard Laminar Flow Hood
(The Baker Co., Sanford, Maine) using sterile technique.
Dissections. The head of each fetus was severed from the body and a single
circumferential cut made just above the eyes. Using fine forceps, the brain was
removed. The tongue was then entirely removed by pushing it through the floor
of the oral cavity with microscissors. As part of the operation, the hole through
which the tongue was removed was enlarged to form a small vent to the outside
to allow greater circulation of the culture medium within the oral cavity.
However, the maxillary-mandibular relationship remained as in vivo (Brinkley,
Basehoar & Avery, 1978). After these operations, fetuses from a given litter
were randomly divided into three groups. Two of the fetuses were fixed immediately to serve as references for the stage of palatal development prior to culture.
Fetuses in the second control group received no further treatment prior to
culture, and thus had intact cranial bases. Those of the third group had the
cranial base disrupted as follows: a mid-sagittal lesion was made in the anteroposterior plane with microscissors and the area extending from approximately
the nasal septum through the craniopharyngeal area was destroyed.
Cranial base and palatal shelf movement
95
Table 1. Numerical system of rating palate closure
Value
assigned
0
1
2
3
4
5
6
Shelf form
Uniform thin ridges of tissue, shelves not fully formed
Vertical along entire length
Anterior quarter vertical, middle quarter partially horizontal, posterior half vertical
Anterior half partially horizontal, posterior half vertical
Anterior quarter partially horizontal, middle half has
the anterior area horizontal, the posterior area partially horizontal, posterior quarter vertical
Anterior horizontal, posterior quarter partially
elevated
Fully horizontal along entire length
Width/length
ratio
1-0-75
075-0-5
0-5-0-25
0-25-0
Contact
Adhesion
Fusion*
* Evidence of epithelial breakdown: shelves appear continuous under the dissecting
microscope, or show epithelial breakdown histologically.
A total of 28 heads with ablated cranial base flexures were randomly selected
and microscopically examined to determine the size of the lesion produced; the
average lesion was found to be 407 ± 110 jam in length (anteroposterior plane)
and 84 ± 26 /im in width.
In the course of devising the surgical techniques to be used in these experiments,
various size lesions were made, including removal of most of the floor of the
cranial vault. In 10 heads as much of the floor of the cranial vault as possible
was removed, leaving only the lateral aspects, nasal capsule and foramen
magnum intact. The palatal shelves of these preparations elevated as well as
control preparations with intact cranial bases. However, as this operation was
difficult to accomplish without damaging other orofacial structures, including
the palatal shelves, the more controlled mid-sagittal ablation was selected as
the standard operation.
Culture. Dissected heads were submerged in the culture chamber by passing
one end of an S-shaped stainless-steel hook through the foramen magnum, and
the other over wires fixed in the chamber. Each head was suspended with the
nose down in the slightly turbulent medium such that palate closure would have
to be accomplished without the aid of gravity. The culture medium, a supplemented Eagle's Basal Medium with 50 % heat inactivated fetal calf serum, was
continually circulated with a peristaltic pump, and oxygenated using hollow
fiber devices. The design of the culture chamber, medium and culture conditions
are reported in detail elsewhere (Brinkley et al. 1975).
Previous work has shown that in fetuses of these ages, in vitro palatal closure
requires 12-16 h (Brinkley et al. 1978). Thus, preparations were cultured 18 h
to provide ample time for shelf movement to take place. At the end of the culture
period the heads were removed from the culture chamber, rinsed in saline
7-2
96
L. L. BRINKLEY AND M. M. VICKERMAN
Table 2. Comparison of mean stages of palatal closure
24 h prior to closure
TimeO
Intact cranial base
Cranial base flexure ablated
18 h prior to closure
TimeO
Intact cranial base
Cranial base flexure ablated
* Different from time 0 values
n
Shelf form
17
38
59
2-47 ±0-62
5-34 + 0-71*
5-59 + 0-72*
4-18 ±1-95
10
16
5-94±0-25*
25
5-64 ± 0-49*
with P — 0-1 using Student's t
Width/length
ratio
20 ±0
408 ±0-78*
413 + 0-73*
2-3 ±0-48
4-38±l-02*
4-2 + 0-91 *
test.
solution and fixed in Bouin's fixative or phosphate buffered formalin. The
mandibles were removed and the state of palate closure assessed by examination
under a dissecting microscope. A numerical system of rating the degree of
palate closure which assigns values to shelf form and width/length ratio was
used (Table 1). 'Width/length ratio' is the ratio of the width of the gap between
the shelves at their closest point to the length of the shelves. This was determined by measuring these two parameters with an ocular micrometer.
Histology. Preparations were fixed in Bouin's fluid, processed and embedded
in paraffin, sectioned at 5 or 10 /tm and stained with hematoxylin and eosin.
RESULTS
All of the midline flexure area was ablated in the lesioned groups (Fig. 1C, D).
However, when mean shelf form and width/length ratio values were compared
(Table 2), no significant difference was found between specimens in the intact
and flexure ablated groups of either age. Heads of both ages, with intact or
ablated cranial base flexures developed to about the same average stage of
palate closure, that is, palatal shelves fully horizontal and contacting by the end
of the culture period. The distribution of width/length ratios relative to shelf
form values was also examined. Using the x2 test, no statistically significant
Fig. 1. A and B are frontal sections of a specimen cultured with an intact cranial
base (20x). Al, Midline region of the adhered palatal shelves indicated by the
arrow in A (280 x). A2, Higher magnification of the same region (560 x). C and D
are frontal sections of a litter-mate with the cranial base flexure ablated prior to
culture (20 x). Cl, Midline region of the adhered palatal shelves indicated by the
arrow in C (280 x). C2, Higher magnification of the same region (560 x). The
palatal shelves of both specimens have moved to the horizontal plane, contacted
and adhered. Epithelial breakdown has begun (A2, C2). The tissues of both
specimens appeared healthy and vital at the end of the 18 h culture period (Al, 2;
Cl, 2). All sections are 10/*m thick and are stained with hematoxylin and eosin.
N, nasal septum; PS, palatal shelf.
Cranial base and palatal shelf movement
97
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L. L. BRINKLEY AND M. M. VICKERMAN
difference in the distribution of shelf forms achieved was noted between intact
and ablated groups for either age.
In the case of the fetuses which were 24 h prior to palate closure achieving
this state required that the entire shelf become horizontal, whereas, in those
which were 18 h prior to palate closure, movement of essentially the anterior
one-quarter and posterior one-half of the shelf was involved.
Histologically, no difference in tissue vitality was observed between preparations with intact or ablated cranial base flexures. The tissues of both types
of preparations appeared healthy with no sign of pycnotic cells at the end of the
culture period (Fig. 1).
DISCUSSION
Verrusio's (1970) mechanical hypothesis of palate closure is based on midline
criteria, thus any change in shape of the midline area would have to be extended
or transmitted laterally to have any effect on the palatal shelves. Long et al.
(1973) also used midline criteria and have proposed that straightening of the
presphenoid is necessary for palatine shelf movement and normal palate closure.
They suggested mitotic division in the midline area as the possible mechanism
of straightening. This suggestion was based on the uptake of [3H]thymidine by
cells of the mid-sagittal area of the craniopharyngeal, presphenoid and
mesethmoid regions over a time period beginning approximately 8 h prior to
closure.
In the present study, various sizes of lesions were made in the cranial base.
These ranged from a mid-sagittal lesion extending posteriorly from approximately the mesethmoid through the craniopharyngeal area to ones in which
virtually the entire floor of the cranial vault was removed. These lesions
destroy the area of presumptive flexure, ablating any potential for form change
in the area far in advance of when straightening is thought to take place. Clearly,
destruction of the so-called pivotal cranial base flexion area had no discernible
negative effects on palatal shelf movement. Thus 'internal shelf force' is
probably not related to the state of the cranial base as suggested by Verrusio
(1970).
If the model of mechanical closure via cranial base straightening were
operating in mice and rats, the cranial base flexure, the key to the operation of
the model, would be expected to be obviously and ubiquitously present in those
species. Several investigators have reported preclosure flexure in the cranial
base of rats and mice (Harris, 1964, 1967; Larsson, 1972; Long et al. 1973;
Diewert, 1976). However, considerable species and strain variation in the
degree of flexure has been noted (Harris, 1964, 1967).
Ablation of the cranial base flexure did not influence palatal elevation in the
present study. A previous study using this system has shown that movement of
the palatal shelves may be blocked by interfering with other craniofacial
structures (Brinkley et al. 1978). Removal of the entire mandible interrupts
Cranial base and palatal shelf movement
99
palatal shelf elevation, whereas removal of only the anterior one-half of the
mandible does not affect it. The findings of the previous study show that palatal
shelf movement in vitro is susceptible to interference by selective alteration of
craniofacial structures. The elevation of the shelves which occurs with an
ablated cranial base demonstrates that the cranial base flexure does not provide
the motive force for elevation in vitro.
Although the mechanical hypothesis tested by the present experiments is
based on observations of the behaviour of a midline cranial base flexure, it is
possible that this flexure is not of local origin, but part of the growth process of
the surrounding craniofacial complex. Craniofacial changes relative to palate
closure have been studied by many investigators (Harris, 1964, 1967; Hart et al.
1972; Wragg, Klein, Steinvorth & Warpeha, 1970; Kriens, 1971; Smiley, Hart
& Dixon, 1971; Schliemann & Kriens, 1971; Larsson, 1972; Taylor & Harris,
1973; Diewert, 1976; Jelinek & Peterka, 1977) and several different parameters
of growth and development have been shown to be changing during the 24 h
preceding closure. The anterior cranial base lengthens and straightens, the
nasal capsule expands and the snout rotates, and the mandible increases in
length.
Our experiments demonstrate that palatal shelf movement is not dependent
upon a midline cranial base flexure. However, we recognize that even in the
most extreme of our experiments small tissue bridges remain anteriorly and
posteriorly. Thus, we cannot rule out the possibility of a flexure of the axis
originating outside the area of the lesion.
This work was supported by U.S.P.H.S. Grant DE-02774, National Institute of Health,
Bethesda, Maryland.
A portion of this work was presented at the meeting of the American Association for
Dental Research, New York, 1975.
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