/ . Embryol. exp. Morph. Vol. 54, pp. 229-240, 1979
Printed in Great Britain © Company of Biologists Limited 1979
229
Elevation of lesioned palatal shelves in vitro
By L. L. BRINKLEY 1 AND M. M. VICKERMAN 1
From the Department of Anatomy, Medical School,
The University of Michigan
SUMMARY
Fetuses were obtained from CD-I mice at a time estimated to be 12 h prior to in vivo secondary palate closure. One of the palatal shelves of each partially dissected fetal head was
lesioned in one of five ways, the other left intact to serve as a control. Single transverse cuts
extending the width of the shelf were made at one of three positions along the longitudinal
axis of the shelf: one-third, one-half or two-thirds the shelf length estimated from the rostral
edge. Some specimens were cut in two places, dividing the shelf into three equal segments.
Another group received a lesion which separated the caudal third of the shelf from its
maxillary connections. All specimens were cultured for 18 h. At the end of the culture period
the heads were fixed, examined and the degree of elevation of each shelf piece assessed.
Intact, contiol shelves of all preparations were elevated in the rostral two-thirds of the
shelf, while the caudal third was partially elevated. Results seen in lesioned shelves depended
upon both the size of the segment and the region of the shelf contained in the segment.
The rostral two-thirds of the shelf, the presumptive hard palate, whether intact or in segments
elevated without physical connections to neighboring shelf tissue. Thus, it is unlikely that
this elevation requires a wave of contraction be transmitted from the caudal soft palate
region. In contrast, the presumptive soft palate requires continuity with the rostral portions
of the shelf both to maintain structural stability and to elevate.
INTRODUCTION
Closure of the secondary palate involves movement of the palatal shelves
from a vertical position on either side of the tongue to a horizontal position
superior to it. Extrinsic factors such as tongue displacement, mandibular
growth, straightening of the cranial base and nonpalatal muscular activity
including mouth opening, swallowing and neck flexion have been cited as
contributing to shelf elevation (Greene & Pratt, 1976). Intrinsic factors have
also been implicated, of which the most important is 'internal shelf force'
(Walker & Fraser, 1956).
The nature of this intrinsic force remains undefined. It was originally postulated to reside in an elastic fiber network (Walker & Fraser, 1956); however,
subsequently no such network was seen in the shelves at the time of palate
closure (Stark & Ehrman, 1958; Loevy, 1962; Frommer, 1968; Frommer &
Monroe, 1968). Other investigators have attributed internal shelf force to
1
Authors' address: Dr L. Brinkley, Department of Anatomy, Medical School, University
of Michigan, Ann Arbor, Michigan 48109, U.S.A.
230
L. L. BRINKLEY AND M. M. VICKERMAN
properties of the extracellular matrix, glycosaminoglycans and collagen (Larsson
1961, 1962; Pratt & King, 1971; Pratt, Goggins, Wilk & King, 1973; Hassell
& Orkin, 1976). Hypotheses and evidence have also been put forth suggesting
that a contractile system generates a vector of force that is transmitted along
the shelf in a caudal to rostral direction (Lessard, Wee & Zimmerman, 1974;
Babiarz, Allenspach & Zimmerman, 1975; Wee, Wolfson & Zimmerman,
1976). Regardless of the origin of shelf force, it is generally acknowledged
that this force may allow the palatal shelves to play an active role in their own
elevation.
Several different observations suggest regional differences in the nature of
internal shelf force. In rodents, the movement of palatal shelves appears to
differ relative to the location along the rostral-caudal axis of the shelf. The
most rostral area of the shelf seems to rotate in an all-or-none manner (Coleman,
1965), whereas remodelling of the caudal area seems to involve a medial
protrusion and lateral regression of shelf tissue (Walker & Fraser, 1956;
Larsson, 1962; Coleman, 1965; Harris, 1967; Pourtois, 1972; Srivastava & Rao,
1979). Whether or not localized internal factors are involved is unknown.
There is also disagreement as to the pattern of shelf movement. Some authors
believe movement starts caudally (posteriorly) and proceeds in a wave-like
manner rostrally (anteriorly) (Walker & Fraser, 1956; Babiarz et al. 1975).
Others suggest that it proceeds from the rostral to the caudal ends of the shelves
(Coleman, 1965; Wragg, Diewert & Klein, 1972). It has also been described
as beginning at the junction of the middle and posterior thirds and then extending both rostrally and caudally (Kochhar & Johnson, 1965). To better understand the intrinsic factors, it is essential that extrinsic factors be controlled. One
way to achieve this control is by using an in vitro system.
The process of palate closure has been shown to take place in an in vitro
system in which many possible extrinsic factors are eliminated (Brinkley,
Basehoar, Branch & Avery, 1975). Neither removal of the tongue and brain
(Brinkley, Basehoar & Avery, 1978) nor ablation of a large midsagittal region
of the cranial base (Brinkley & Vickerman, 1978) interferes with in vitro palate
closure. In vitro it is possible directly to observe and manipulate the palatal
shelves themselves with little or no interference from other extrinsic anatomical
structures.
The present study on palatal shelf behavior, using our in vitro system,
investigates two questions: (1) will elevation occur if the longitudinal axis of
the shelf is surgically interrupted, thus determining if transmission of a force
along the longitudinal axis of the shelf is required; and (2) do different areas
of the palatal shelf differ in their ability to elevate when freed from their
attachment to the rest of the palatal shelf. If such differences exist it would
suggest a possible regionalization of whatever factors constitute internal shelf
force.
Lesioned shelf elevation in vitro
231
MATERIALS AND METHODS
Animals. Timed pregnant CD-I mice were obtained from Charles River
Laboratories (Charles River, Massachusetts, U.S.A.). The pregnant mice were
killed by cervical dislocation on late day 13 of gestation (plug day = 0), at a
time estimated to be about 12 h prior to expected palate closure. Procedures
for obtaining the fetuses have been described elsewhere (Brinkley et al. 1975).
As there is variation in time of fertilization of these commercially supplied
mice, as well as developmental variation within a litter, the age of each fetus
was judged on gestational age, crown-rump length and morphological criteria.
Morphological criteria included assignment of a numerical value to the developmental status of forefeet, hindfeet, ears, hair follicles and eyes as described by
Walker & Crain (1960). Fetuses of morphological rating 7-8, with a crownrump length of 10-5-10-7 mm were used for this study. Palate closure occurs
at about 14 days, 8-12 h in CD-I mice at a time when fetuses show a morphological rating of 9-12 and a crown-rump length of approximately 12-5 mm.
Dissections. Fetuses were removed from their membranes and placed in
4 °C culture medium for the remainder of the dissection. This and all subsequent operations were carried out in an Edgegard Laminar Flow Hood
(The Baker Co., Sanford, Maine) using sterile technique. With the aid of a
dissecting microscope, the head of each fetus was severed from the body and
a single circumferential cut around the head was made just above the eyes.
This severed tissue and the entire brain and any attached spinal cord was
removed with fine forceps. The tongue was then entirely removed by pushing
it through the floor of the oral cavity with microscissors. The hole through
which the tongue was removed was enlarged to form a small vent to the outside.
This vent provides both increased medium circulation within the oral cavity,
and access to the shelves for lesioning while leaving the maxillary-mandibular
relationship intact. Dissection procedures have been previously shown to be
the optimal ones for palate closure in this culture system (Brinkley et al. 1978).
One of the pair of palatal shelves of each of 103 fetuses was selectively cut
at various sites along the rostral-caudal axis of the shelf using microscissors
or an ophthalmological scalpel. The other was left intact. Shelves were given a
single transverse cut across the entire shelf at approximately one-third, one-half
and two-thirds of shelf length, or at both one-third and two-thirds of the
length (Table 1). The width of the lesions ranged from 50 to 100/tm. In an
additional 14 specimens the lateral (maxillary) connections of the caudal
one-third of one shelf were severed (Fig. 1A).
The fetal rat palate just prior to closure is assumed to be composed of
approximately two-thirds presumptive hard palate, divisible anatomically into
anterior, middle and posterior regions, and one-third soft palate (Diewert,
1978). This assumption was verified for the mice used in the present experiment.
Ten heads were examined to determine the approximate proportion of the total
232
L. L. BRINKLEY AND M. M. VICKERMAN
Table 1. Relationship of the site of a transverse lesion
to shelfform after culture
Shell" form after culture
AH*
Mil
nr
Control shelves (n = 103)
s
100%
Single Lesion: f
100%
(1) At-J-shelf length (« = 20)
55%
(2) At+shelf length (//. = 29)
100%
(3) Atfshelf length (« = 23)
Two Lesions
(4) At-^ and 3-shelf length
1
mm
mm*
77%
16%
6%
Horizontal,
Partially elevated,
Vertical.
* Postulated anatomical regions of the shelf: All = anterior hard palate: Mil = middle
hard palate; I'll = posterior hard palate; S = soft palate.
t The placement of all lesions is described using the rostral edge of the palate as the reference point.
Lesioned shelf elevation in vitro
233
shelf length which could be assigned to each of these four regions, using the
anatomical criteria of Diewert (1978). The anterior and middle hard palate
represented approximately 20 % each of the overall length, the posterior hard
palate and soft palate, 30% each as shown in the schematic diagrams in
Table 1.
Culture. The preparations were suspended and immersed in circulating,
gassed culture medium as previously described (Brinkley et al., 1975). A
modification in the design of the chamber has been made which enhances
circulation of medium and gas exchange (Lewis, Thibault, Pratt & Brinkley,
1979). An 18 h culture period was selected to minimize the time available for
wound healing, while providing sufficient time for elevation to occur in this
system (Brinkley et al. 1978). Commercially supplied BGJb medium (Grand
Island Biological Co., Grand Island, N.Y., U.S.A.) with an additional 0-6 mg/ml
glutamine and 50/ig/ml gentamicin, supplemented with 10% heat-inactivated
fetal calf serum (KC Biological, Lenexa, Kansas, U.S.A.) was used. The
medium was circulated at a rate of 10 ml/min, gassed with 95 % O2, 5 % CO2
using silicone co-polymer follow fiber devices (NIH, Bioengineering Laboratory,
Bethesda, Maryland, U.S.A.), and kept at 34 °C. The P O a and P C o 2 values of
the medium were determined with an IL 113 Blood Gas Analyzer. The average
P Oa was 525 mmHg, P C Q 2 50 mmHg.
Assessment of shelf form. After culture, the heads were removed, fixed in
Bouin's fluid or phosphate-buffered formalin and the mandible removed. With
the aid of a dissecting microscope, each portion of the lesioned shelf, as well as
the intact control shelf, was assessed for its degree of elevation and for its
alignment relative to the intact shelf. Fifty-eight preparations were embedded
in paraffin or methacrylate and sectioned to determine the vitality of the tissue
surrounding the lesions.
RESULTS
Intact shelves
The intact, control shelves of all cultured specimens were elevated in the
rostral two-thirds of the shelf, while the caudal third ranged from partially to
fully elevated. As a majority of the caudal thirds were only partially elevated,
the control form of this region is reported as partially elevated. These results
are similar to those seen in fetuses of this age that have both shelves intact
(Lewis et al. 1979).
Site of the lesions and shelf form after culture
The relationship between the site of the lesion and the form exhibited by
experimental shelves at the end of the culture period is shown in Table 1.
With a single cut placed at the junction of the rostral one-third and caudal
two-thirds of the palatal shelf (lesion 1) both segments achieved the same shelf
form as that of the intact control shelves. However, with a single cut placed
234
L. L. BRINKLEY AND M. M. VICKERMAN
Fig. 1. Photogiaphs of the palatal region of two lesioned specimen after 18 h culture.
(A) The caudal third of the shelf has been severed from its lateral maxillary connections. The arrows indicate the area and approximate extent of the lesion. The
caudal region of the lesioned shelf has retained its form and partially elevated.
(B) One shelf has been lesioned at approximately one-third and two-thirds the shelf
length. The rostral (R) and intermediate (I) thirds have elevated and adhered,
whereas the caadal third (C) has moved laterally and has not maintained normal
form.
at two-thirds the shelf length (lesion 3), only the large rostral segment appeared
similar to controls; whereas the caudal third showed no form change. When the
shelf was bisected (lesion 2) the rostral one-half achieved control form in 90 %
of the cases, whereas the caudal piece did so only 65 % of the time. When the
shelf was divided into three approximtely equal segments the shelf form
assumed by a given piece was directly related to its location along the longitudinal axis of the shelf. The rostral and intermediate segments had the same
form as equivalent regions of an intact shelf in 77 % and 93 % of the cases,
respectively, whereas the caudal third never did.
Influence of segment size and region on shelf form
When the data shown in Table 1 are examined by segment size and shelf
region the influence of these two factors can be seen. All of the hard palate
regions were more stable than the soft palate. That is, isolated segments of
hard palate behaved alone as they would when in an intact shelf. However, this
was not true of the soft palate as is seen by the variation in form achieved by
shelf thirds. Form similar to controls was achieved by all the intermediate
thirds, composed principally of posterior hard palate, 86 % of rostral thirds,
principally anterior hard palate, and no caudal thirds, soft palate. In addition
the caudal thirds also lost normal form by moving laterally and becoming
rounded (Fig. 1B). Since no piece was predominantly composed of the middle
Lesioned shelf elevation in vitro
235
hard palate, no evaluation of its form change when alone was possible. However, middle hard palate composed approximately 40% of rostral thirds and
20% of intermediate thirds. In 90% of these segments control shelf form was
observed.
Rostral halves of palatal shelves, composed entirely of hard palate, achieved
form similar to controls in 90% of the cases. On the other hand, caudal halves
which included more than half (ca. 60%) soft palate, behaved similarly to
controls only 66 % of the time. The dampening influence of the soft palate was
not seen, however, in the behavior of the caudal two-thirds segments which
were composed of approximately equal parts hard and soft palate. In this
case these regions behaved similarly to the same regions of intact shelves in
all cases.
Another type of cut was made to determine more about the behavior of the
caudal area. In 14 specimens the posterior one-third of the shelf was freed
from its lateral maxillary connections, but remained attached to the rostral area
of the shelf. In all cases the piece retained recognizable shelf form and achieved
the partially elevated control shelf form (Fig. 1A).
Misalignment
In addition to movement to the horizontal plane, the shelves must align with
one another in such a way that the corresponding areas along their longitudinal
axes are in register. When two intact shelves elevate this occurs naturally;
however, when examining the position of shelf segments, significant misalignment was found to occur in pieces of less than or equal to half the shelf length.
Rostral halves were misaligned in 68 % of the cases, whereas caudal pieces were
misaligned in 42% of the cases. The proportion of misalignment increased
dramatically when the shelf was divided into three pieces. All of the caudal
thirds showed a loss of form. The caudal piece no longer had recognizable shelf
contours and in all cases moved laterally from its original position (Fig. 1B).
The rostral thirds maintained normal shelf contours, but were misaligned in
53 % of the cases. In sharp contrast, the intermediate thirds were not found to
be misaligned on observation with the dissecting microscope.
Histological findings
Fifty-eight specimens were examined histologically to determine tissue vitality
and to note any evidence of wound healing. Figure 2 illustrates sections immediately surrounding and within a lesion. Such tissue appeared vital, despite
the presence of some dead cells in the wound area itself. No evidence of wound
healing was observed.
16
EMB
54
236
L. L. BRINKLEY AND M. M. VICKERMAN
Fig. 2. Coronal sections illustrating shelf behavior and tissue vitality in the regions
immediately surrounding a typical transverse lesion made at approximately onethird the shelf length. The shelf tissue surrounding the lesion has maintained form,
elevated and adhered. (A) Central area of the lesion illustrating its lateral extent.
(B) Posterior area of the lesion. (C) Immediately caudal to the lesion. The shelf is
again intact and in contact with the control shelf. (D-F) Higher magnification of
the tissue immediately adjacent to the lesion. In all sections the cells appear vital,
with no evidence of wound healing. Section thickness, 3/«n; stain, toluidine blue.
N = nasal septum; PS = palatal shelf.
DISCUSSION
The ability to directly manipulate palatal shelves and follow the outcome
in vitro provides a unique opportunity to gain information on the stability of
form and elevation behavior of palatal shelf regions. Our results indicate that
elevation of the rostral two-thirds of the shelf, the presumptive hard palate,
is not dependent on elevation of the caudal third, the soft palate. This is in
agreement with the observations of Walker & Quarles (1976) that shelf elevation
occurred rostrally in fetuses with tongue stubs which prevented caudal shelf
Lesioned shelf elevation in vitro
237
elevation. Present results also demonstrate that the hard palate itself need not
be intact, since segments of presumptive hard palate which are approximately
one-third the total shelf length will elevate. This clearly indicates that elevation
of the hard palate does not require that a vector of force be transmitted from
the soft palate, thus arguing against the necessity of a 'wave of elevation' or
vector of force emanating from the caudal area (Walker & Fraser, 1956;
Babiarz et al. 1975; Wee et al. 1976).
Rather than being uniform throughout their length, the palatal shelves display
a striking regionalization of both elevation behavior and stability of form. The
hard palate, particularly the posterior region, displayed an inherent ability to
assume the horizontal position even in segments composed of only one-third
the total shelf length. In addition, the posterior hard palate was never found to
be misaligned, and therefore seems to be the most structurally stable region.
It would seem functionally advantageous for elevation to begin at the most
stable region of the shelf, namely the middle third or presumptive posterior
hard palate. This is supported by the observations of Kochhar & Johnson (1965)
that palatal shelf movement in rats is initiated at the junction of the anterior
two-thirds and the posterior one-third of the shelves. We have also observed
this in mice during in vitro palate closure (unpublished observation).
The behavior of the caudal, presumptive soft palate region of the shelf
contrasts sharply to that of the hard palate. Not only is the caudal third unable
to elevate when it is isolated from the rest of the shelf, but it does not retain
its normal form, as do the regions of the hard palate. Caudal thirds lose their
form by appearing to move or collapse laterally. Skeletal muscle fibers reportedly
insert from the maxillary area into the buccal side of the most caudal region
of the shelf (Babiarz et al. 1975). The lateral movement of this region when it
is freed from most of its connections with the more morphologically stable hard
palate, may reflect contractile actions of these skeletal muscle fibers. Some
support for this view comes from our observation that the caudal region of the
shelf will elevate when severed from its maxillary connections.
Another factor in the failure of the caudal third to elevate may be the local
disruption of epithelial adhesion at the medial edge caused by the experimental
transverse cuts. That is, a part of caudal elevation may be dependent on a
'zippering' effect occurring as the medial edge adhesion of the shelves progresses
rostrally and caudally. From our observations it appears that the elevation
force for the soft palate must be coming in part from its connections with the
hard palate. Whether this is merely supplying structural stability, or is, in fact,
supplying some sort of motive force is unknown.
The primary experimental procedure in the present study, making a transverse
cut perpendicular to the long axis of the shelf, disrupts the integrity of the
epithelial covering in that local area, destroys underlying mesenchymal cells in
the path of the lesion, and disrupts the continuity of any elements which are
disposed along the long axis of the shelf. However such lesions do not, except
16-2
238
L. L. BRINKLEY AND M. M. VICKERMAN
for the localized point of the lesion, affect the maxillary attachments of the
shelves. Whatever the intrinsic elements creating the motive force for shelf
elevation, they are presumably distributed throughout the hard palate, at least,
and are not dependent on the integrity of the whole shelf for their function.
Thus, it is logical to look at the structural components of the shelf, the epithelial
cells, mesenchymal cells and extracellular matrix, as the potential basis of
intrinsic shelf force. In that regard many proposed roles of these components
are consistent with the findings of the present study.
The epithelial cells encasing the mesenchymal and extracellular matrix components may provide part of the shelf force in one of several ways. The surface
area of the shelf epithelium may be decreased on the nasal (lingual) surface by
an increased adhesiveness between epithelial cells. Also, if the surface area of
the oral epithelium remains constant the formation of rugae could reduce the
overall dimensions of the buccal epithelium, mostly in the rostral-caudal
direction. It has been suggested that these two epithelial activities acting
together could cause a bulging of the shelf tissue toward the nasal slope (Pourtois,
1972).
Many investigators have also suggested that networks or regions of concentration of mesenchymal cells exist which may play a role in shelf elevation
(Walker, 1961; Larsson, 1962; Kochhar & Johnson, 1965; Pourtois, 1972;
Babiarz et al. 1975; Wee et al. 1976). For instance, preosteoblastic masses of
mesenchymal cells found in the lateral maxillary areas could act as anchors for
other contractile mesenchymal cells or convey tension by some other means
(Pourtois, 1972; Babiarz et al. 1975).
Extracellular components of the shelf may also be important. Several workers
have demonstrated the presence of glycosaminoglycans throughout rodent
palatal shelves near the time of closure, and have suggested that these molecules,
especially hyaluronate, are important in shelf elevation (Larsson, 1960, 1961,
1962; Walker, 1961; Kochhar & Johnson, 1965; Pratt et al. 1973; Ferguson,
1978; Wilk, King & Pratt, 1978). Evidence from qualitative histochemistry
indicates that the middle third of the shelf, or the posterior hard palate,
appears to contain the most extracellular matrix (Kochhar & Johnson, 1965).
Collagen fibrils have also been reported to be present adjacent to the basement
membrane of the oral epithelium, oriented in a parallel array along the rostralcaudal axis of the shelf (Hassell & Orkin, 1976). However, the cuts used in the
present study would be expected to disrupt this longitudinal array.
Thus, it seems that elevation of the presumptive hard palate may actually
be the expression of a pre-existing infrastructure. That is, when the shelves are
no longer held in a vertical position by the tongue, they assume a predetermined
form built into them by the quality, quantity and distribution of the cells,
extracellular matrix and collagen within the shelves and adjacent maxillary
area. In contrast, when the soft palate is not part of an intact shelf it does not
seem to have sufficient internal stability to maintain its form and elevate.
Lesioned shelf elevation in vitro
239
This work was supported by U.S.P.H.S. Grant DE-02774, National Institutes of Health,
Bethesda, Maryland, U.S.A.
A portion of this work was presented at the meeting of the International Association of
Dental Research, Copenhagen, Denmark, 1977.
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{Received 9 May 1979, revised 16 June 1979)