ORIGINAL ARTICLE

Functional inuence on sutural bone apposition

in the growing rat
Christos Katsaros,
a
Alexandros Zissis,
b
Andrea Bresin,
c
and Stavros Kiliaridis
d
Gteborg, Sweden, Nijmegen, The Netherlands, Saarbrcken, Germany, and Geneva, Switzerland
Introduction: The aim of this study was to quantify the inuence of reduced masticatory muscle function on
sutural bone apposition in the growing rat. Methods: Twenty-six growing male albino rats were randomly
divided into 2 equal groups; 1 (hard-diet group) received the ordinary diet of hard pellets, and the other
(soft-diet group) received the powdered form of the ordinary diet mixed with water. The experimental period
started when the rats were 4 weeks old and lasted 42 days. At days 0, 14, and 28, calcein was injected into
all animals. At the end of the experiment, the animals were killed, and the heads were taken for preparation
of undecalcied frontal sections, 120 m thick. Three representative homologous sections for each animal
in both groups were selected and studied under a uorescence microscope. The level of bone apposition at
the time of calcein injection was marked with separate uorescing lines. Because the lines from the rst
injection could not be seen in all areas, bone apposition in the internasal, naso-premaxillary, and
inter-premaxillary sutures was quantied from day 14 to the end of the experimental period, by using an
image analysis software. Results: In both groups, greater bone apposition was found between days 14 and
28 than between days 28 and 42 of the experimental period. Less bone apposition was found in the soft-diet
group than in the hard-diet group in all sutures studied. Conclusions: The ndings suggest that bone
apposition in the studied facial sutures in the anterior facial skeleton of the growing rat is signicantly affected
by reduced masticatory function. (Am J Orthod Dentofacial Orthop 2006;129:352-7)
A
lthough various experimental and clinical studies
have shown associations between masticatory
muscle function and craniofacial growth,
1,2
the
response of certain growth sites such as sutures is not fully
elucidated.
Mechanical loading seems to play an important role
in the regulation of sutural growth.
3-5
Also, the devel-
opment of interdigitations in the sutures seems to be
related to the functional strains placed on a tissue.
6-13
Moreover, in histological studies, morphological differ-
ences in the sutures in the anterior part of the snout and
a number of synostoses in the internasal suture were
observed in rats with reduced masticatory function,
possibly because there was less tension on the premax-
illa and the lateral part of the nasal bones.
14,15
The reduction of the loads applied to the skull
during mastication by feeding the animals a soft diet
was found to adversely inuence the transverse growth
of the premaxilla at areas under direct muscle inuence
near their attachments.
16
It is not fully elucidated
whether the narrower internasal, naso-premaxillary,
and inter-premaxillary sutures and the more parallel
orientation of the bony surfaces of the naso-premaxil-
lary suture under reduced functional demands reported
in a previous study
17
are related to decreased sutural
bone apposition and thus contribute to the above-
mentioned reduction in the width of the premaxilla.
The aim of this study was to quantify the inu-
ence of reduced masticatory muscle function on
sutural bone apposition in the anterior facial skeleton
of the growing rat.
MATERIAL AND METHODS
Twenty-six growing male albino rats of the
Sprague-Dawley strain, with a mean body weight of
100 g at the beginning of the experiment, were used.
They were housed 4 or 5 in each cage and were fed and
watered ad libitum. The experimental protocol was
approved by the Ethics Committee of Gteborg Uni-
versity. The animals were randomly separated into 2
equal groups (n 13) according to diet; 1 (hard-diet
a
Professor, Department of Orthodontics and Oral Biology, Radboud University
Nijmegen Medical Center, Nijmegen, The Netherlands; former research asso-
ciate, Department of Orthodontics, Gteborg University, Gteborg, Sweden.
b
Private practice, Saarbrcken, Germany.
c
Former research associate, Department of Orthodontics, Gteborg University,
Gteborg, Sweden.
d
Professor, Department of Orthodontics, University of Geneva, Geneva,
Switzerland.
Supported by grants from the Swedish Medical Research Council (05006).
Reprint requests to: Professor C. Katsaros, Department of Orthodontics and
Oral Biology, Radboud University Nijmegen Medical Center, 309 Tand-
heelkunde, PO Box 9101, 6500 HB Nijmegen, The Netherlands; e-mail,
C.Katsaros@dent.umcn.nl.
Submitted, April 2003; revised and accepted, September 2004.
0889-5406/$32.00
Copyright 2006 by the American Association of Orthodontists.
doi:10.1016/j.ajodo.2004.09.031
352
group) received the ordinary diet of hard pellets (Diet R
47, Astra Ewos A B, Sdertalje, Sweden), and the other
(soft-diet group) received the powdered form of the
ordinary diet mixed with water in standardized propor-
tions (2:5, R 47 and water).
18
The experimental period started when the rats
were 4 weeks old and lasted 42 days; all rats were
weighed 3 times a week. On days 0, 14, and 28 of the
experimental period, calcein was injected into all
animals (1.5 mg per 80 g).
After the rats were killed, their heads were taken for
preparation of undecalcied sections. The specimens
were xed in 10% buffered formalin, dehydrated in
alcohol of progressively increasing concentrations, and
embedded in methylmethacrylate.
19
Frontal sections
120 mm thick were prepared with a Leitz-Saw mic-
rotome.
To study homologous representative sections from
each animal, the last section before the anterior palatine
foramen was taken as a common basis for selection.
Then 2 sections were selected 1.6 mm apart mesially
(Fig 1). This selection was made because the previously
reported differences in the width of the facial sutures
under different functional demands were found mostly
in this part of the snout.
17
The selected sections were viewed with an Olym-
pus BHS uorescence microscope (objective Olympus
SPLAN FL2). An excitation lter and barrier lters
(Olympus 20BP490, 17AFC, and 17O515) were used
to obtain light at wavelengths of 435-490 nm. The level
of bone apposition at the 3 times of calcein injection
was marked with separate uorescing lines (Fig 2).
Because the lines from the rst injection could not be
seen in all areas because the bone had resorbed or the
uorescing lines were weak, bone apposition was
quantied from day 14 to the end of the experimental
period.
Optical recording was done with a Sony DXC-
LS1P CCD color video camera on an Olympus U-
PMTVC camera adapter. The images were loaded into
a computer (Intel Pentium II 350 PC, 128 MB, Win-
dows NT4.0 WS SP2) by means of a frame grabber
(Logitech Snappy) at a resolution of 1500 1125
pixels and 24-bit color resolution. The images were
then checked for measurability and motion artifacts by
using Photoshop 4.0 (Adobe) and stored as uncom-
pressed TIFF les for further processing. For calibra-
tion purposes, a 100-m scale was also recorded in the
same way as the sections.
Image analysis was performed on a separate com-
puter (AMD K6-2 500, 128MB, Linux 2.2.14,
Xfree86 3.3.6) by using the free software GIMP 1.0
(GNU Image Processor). The resulting data were re-
corded and processed with the spread sheet application
StarCalc from the StarOfce Suite V5.1a (SUN Micro-
Fig 1. a, Ventral (left) and lateral (right) view of rat skull. Vertical lines mark 3 sections used. Longer
line denotes section taken as common basis for selection; b, schematic illustration of frontal section
of rat skull (base section). IN, Internasal suture, NP, naso-premaxillary sutures; IP, inter-premaxillary
suture.
American Journal of Orthodontics and Dentofacial Orthopedics
Volume 129, Number 3
Katsaros et al 353
systems) on the same machine, with Microsoft Excel
Data format for transportability.
First, the scale image was measured, giving a
resolution of 520 pixels/mm, so that subsequent calcu-
lations were performed by using 0.52 pixels/m as a
conversion factor. The internasal, naso-premaxillary,
and inter-premaxillary sutures were studied. The bone
apposition was measured at 5 approximately equidis-
tant locations distributed over the length of each suture
perpendicular to the bone apposition sites. Measure-
ments were made from the day-14 line to the day-28
line, and from the day-14 line to the nal bone level at
the sutural edges.
The sutural bone apposition was to be presented as
the mean of the 2 sutural edges. For that purpose, the
mean of all measurements was calculated for each
suture at each section. This calculation method was
used to compensate for any differences in the way the
tissue blocks were sectioned (angle between saw and
long axis of the skull).
For each rat, the mean of all 3 sections was used.
The Student t test was used to test for differences
between the hard-diet and the soft-diet groups. To
compensate for differences in growth, sutural bone
apposition between days 14 and 42 was also compared
between the 2 groups after correction for nal weight
and nose-tail distance (at day 42) by using analysis of
covariance (ANCOVA). Paired t tests were used for
comparison between the different sutures.
Double measurements on 20 sections were made
to evaluate the method error. The magnitude of the
combined method error was calculated according to
Dahlbergs formula: Se d
2
/2n, where d is the
difference between 2 registrations of a pair, and n is the
number of the double registrations.
20
The average
combined method error was found to be 2.3 m. Paired
t tests were used between the 2 series of measurements
to test for systematic errors; no statistically signicant
differences were found.
RESULTS
The rats mean body weights, weight gains, and
nose-tail distances were similar in the 2 groups. In the
hard-diet group, however, there were outliers for nal
mean body weight (at day 42) and weight gain (Table I).
In both groups, greater bone apposition was found
between days 14 and 28 than between days 28 and 42
of the experimental period (Table II).
The bone apposition rate was found to be lower in
the soft-diet group than in the hard-diet group in all
sutures studied (Table II). Only in the inter-premaxil-
lary suture was the difference between days 14 and 28
not statistically signicant. Especially in the naso-
premaxillary suture, bone apposition between days 14
and 42 was 26% less in the soft-diet group than in the
hard-diet group. Also, after the correction for nal
weight and nose-tail distance, the magnitude of the
differences in bone apposition between the groups did
not change (Table III).
The amount of bone apposition differed between
the sutures in both groups. The greatest bone apposition
was registered in the nasal side of the naso-premaxil-
lary suture, followed by the inter-premaxillary suture
on both sides. The internasal suture showed the least
bone apposition (Table II). The differences between the
sutures were statistically signicant (P .0001).
DISCUSSION
In this study, the inuence of masticatory muscle
function on sutural bone apposition was investigated by
reducing the loads applied to the skull during mastica-
tion. This reduction was experimentally induced by
feeding the animals a soft diet. This experimental
model enables the effects of an altered function to be
observed in an intact masticatory system.
1
Fig 2. Undecalcied section of interpremaxillary suture
under uorescent microscopy. Days 0, 14, and 28:
uorescing lines marking level of bone apposition at
calcein injection. Day 42: bone surface at sutural edge.
Fluorescing line at day 0 is weak and missing in some
parts of suture because that bone has been resorbed.
American Journal of Orthodontics and Dentofacial Orthopedics
March 2006
354 Katsaros et al
Our ndings suggest that reduced masticatory mus-
cle function results in decreased bone apposition rate in
the plane facial sutures of the growing rat. The fact that
the differences in bone apposition between the 2 groups
were still present after correction for nal weight and
nose-tail distance shows that the bone apposition rate
was not inuenced by intergroup differences in overall
growth. Thus, the differences in bone apposition be-
tween the 2 groups could be attributed to the effect of
the altered masticatory muscle function.
The differences in the amount of bone apposition
between the sutures show the different modeling
pattern in the various regions of the facial skeleton.
In the naso-premaxillary suture, most of the bone
apposition occurred at the nasal part, whereas the
premaxillary part showed weak apposition. In con-
trast, cortical bone apposition is much higher in the
upper part of the premaxilla than in the nasal bone.
These regional differences in bone apposition are
important in accommodating the changes in form and
Table I. Means and standard deviations (SD) of weight and nose-tail distance of rats and signicance of intergroup
differences
Hard-diet group
(n 13)
Soft-diet group
(n 13)
Signicance Mean SD Mean SD
Weight (g) (day 0) 118.6 5.7 120.5 5.7 NS
Weight (g) (day 42) 393.1 61.5* 370.7 18.0 NS
Weight gain (g) (days 42-0) 274.5 61.1* 250.2 19.8 NS
Nose-tail distance (mm) (day 42) 44.7 1.8 44.1 1.3 NS
*1 outlier: weight 558.0 g; weight gain 436.0 g.
NS, not signicant.
Table II. Means and standard deviations (SD) of overall bone apposition, based on all sections, with signicance of
intergroup differences
Bone apposition (m)
Hard-diet group
(n 13)
Soft-diet group
(n 13)
Signicance (P value) Mean SD Mean SD
Internasal (14-28) 42.6 4.1 36.2 3.6 .0005
Internasal (14-42) 77.5 6.6 63.8 5.4 .0005
(14-28/14-42) x % 55.0 56.7
Naso-premaxillary (14-28) 88.1 9.0 66.5 11.3 .0005
Naso-premaxillary (14-42) 157.5 16.4 116.3 21.1 .0005
(14-28/14-42) x % 55.9 57.2
Inter-premaxillary (14-28) 57.1 5.8 56.0 6.6 NS
Inter-premaxillary (14-42) 103.3 10.7 93.3 11.1 .03
(14-28/14-42) x % 55.3 60.0
(14-28/14-42) x %, Ratio of bone apposition between days 14-28 and days 14-42 expressed as percentage.
NS, Not signicant.
Table III. Differences in bone apposition between hard-diet and soft-diet groups for nal weight and nose-tail distance
(day 42)
Without
correction After correction
Mean SE Signicance (P value) Mean SE Signicance (P value)
Internasal (14-42) 13.7 2.4 .00005 12.7 2.4 .00005
Naso-premaxillary (14-42) 41.2 7.4 .00005 39.4 7.8 .00005
Inter-premaxillary (14-42) 10.0 4.3 .04 9.2 4.5 .05
SE, Standard error of mean.
American Journal of Orthodontics and Dentofacial Orthopedics
Volume 129, Number 3
Katsaros et al 355
spatial orientation of the nasal and premaxillary
bones during growth.
The decreased bone apposition rate under reduced
functional demands might be related to the decreased
width of the sutural space recorded in a previous study
with a comparable experimental design.
17
The smaller
separation of the sutures under reduced functional
demands might be the result of decreased tension in the
periosteal membrane in the suture because of lower
contractive forces of the elevator muscles. This can be
explained by the alteration in the development of the
elevator muscle ber size and composition
21
as well as
their contractive capacity
22
because of the prolonged
alteration of masticatory function. The tension in the
periosteal membrane has been shown to regulate the
displacement of bones in the craniofacial region.
23,24
The ndings of Behrents et al,
25
who showed experi-
mentally in living monkeys that bite forces could
provide sufcient tensile strain to cause separation of
the sagittal suture of the skull, support this aspect.
The decreased sutural bone apposition rate might
have contributed to the decreased width of the premax-
illa under reduced functional demands at areas under
direct muscle inuence near the site of their attach-
ment.
16
The decrease in the contractive forces of the eleva-
tor muscles after reduced masticatory muscle function
also results in decreased occlusal loading, which can
inuence growth even in regions without muscle at-
tachment. The smaller width and the lower ossication
rate of the synchondrosis of the midpalatal suture found
in rats fed a liquid diet support this aspect.
26
Our ndings differ from those on cranial sutures in
miniature pigs when the magnitude of sutural strain was
not correlated with sutural bone apposition rate.
13
However, this either could reect the anatomical and
functional differences between facial and cranial su-
tures or might be due to the anatomical differences
between the 2 species. A rats snout is positioned much
more anteriorly relative to the posterior occlusion than
a miniature pigs snout, and thus the biomechanics of
the facial skeleton during mastication are probably
different. Moreover, in the rat, the anterior and middle
parts of the internasal suture are butt-ended, whereas
they are interdigitated in the miniature pig. That could
imply that tensile strains are applied during mastication
in the rat and compressive strains in the miniature
pig.
10,11,13,27
CONCLUSIONS
Our ndings suggest that the bone apposition rate in
the facial sutures in the anterior facial skeleton of the
growing rat is signicantly affected by reduced masti-
catory function. This might be a mechanism responsi-
ble for osteogenesis in the facial sutures.
We thank Stina Olsson, University of Gteborg, and
Frank Sick, University of Saarland, for their technical
assistance and Professor Martin vant Hof, biostatistician,
University of Nijmegen, for his statistical advice.
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Editors of the International Journal of Orthodontia (1915-1918),
International Journal of Orthodontia & Oral Surgery (1919-1921),
International Journal of Orthodontia, Oral Surgery and Radiography (1922-1932),
International Journal of Orthodontia and Dentistry of Children (1933-1935),
International Journal of Orthodontics and Oral Surgery (1936-1937), American
Journal of Orthodontics and Oral Surgery (1938-1947), American Journal of
Orthodontics (1948-1986), and American Journal of Orthodontics and Dentofacial
Orthopedics (1986-present)
1915 to 1932 Martin Dewey
1931 to 1968 H. C. Pollock
1968 to 1978 B. F. Dewel
1978 to 1985 Wayne G. Watson
1985 to 2000 Thomas M. Graber
2000 to present David L. Turpin
American Journal of Orthodontics and Dentofacial Orthopedics
Volume 129, Number 3
Katsaros et al 357