ORIGINAL ARTICLE

Functional influence on sutural bone appositionin the growing ratChristos Katsaros,a Alexandros Zissis,b Andrea Bresin,c and Stavros Kiliaridisd

Göteborg, Sweden, Nijmegen, The Netherlands, Saarbrücken, Germany, and Geneva, Switzerland

Introduction: The aim of this study was to quantify the influence of reduced masticatory muscle function onsutural bone apposition in the growing rat. Methods: Twenty-six growing male albino rats were randomlydivided 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 periodstarted when the rats were 4 weeks old and lasted 42 days. At days 0, 14, and 28, calcein was injected intoall animals. At the end of the experiment, the animals were killed, and the heads were taken for preparationof undecalcified frontal sections, 120 �m thick. Three representative homologous sections for each animalin both groups were selected and studied under a fluorescence microscope. The level of bone apposition atthe time of calcein injection was marked with separate fluorescing lines. Because the lines from the firstinjection could not be seen in all areas, bone apposition in the internasal, naso-premaxillary, andinter-premaxillary sutures was quantified from day 14 to the end of the experimental period, by using animage analysis software. Results: In both groups, greater bone apposition was found between days 14 and28 than between days 28 and 42 of the experimental period. Less bone apposition was found in the soft-dietgroup than in the hard-diet group in all sutures studied. Conclusions: The findings suggest that boneapposition in the studied facial sutures in the anterior facial skeleton of the growing rat is significantly affected

by reduced masticatory function. (Am J Orthod Dentofacial Orthop 2006;129:352-7)

Although various experimental and clinical studieshave shown associations between masticatorymuscle function and craniofacial growth,1,2 the

response of certain growth sites such as sutures is not fullyelucidated.

Mechanical loading seems to play an important rolein the regulation of sutural growth.3-5 Also, the devel-opment of interdigitations in the sutures seems to berelated 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 anda number of synostoses in the internasal suture wereobserved in rats with reduced masticatory function,

aProfessor, Department of Orthodontics and Oral Biology, Radboud UniversityNijmegen Medical Center, Nijmegen, The Netherlands; former research asso-ciate, Department of Orthodontics, Göteborg University, Göteborg, Sweden.bPrivate practice, Saarbrücken, Germany.cFormer research associate, Department of Orthodontics, Göteborg University,Göteborg, Sweden.dProfessor, 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 andOral 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/$32.00Copyright © 2006 by the American Association of Orthodontists.

doi:10.1016/j.ajodo.2004.09.031

352

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 skullduring mastication by feeding the animals a soft dietwas found to adversely influence the transverse growthof the premaxilla at areas under direct muscle influencenear their attachments.16 It is not fully elucidatedwhether the narrower internasal, naso-premaxillary,and inter-premaxillary sutures and the more parallelorientation of the bony surfaces of the naso-premaxil-lary suture under reduced functional demands reportedin a previous study17 are related to decreased suturalbone apposition and thus contribute to the above-mentioned reduction in the width of the premaxilla.

The aim of this study was to quantify the influ-ence of reduced masticatory muscle function onsutural bone apposition in the anterior facial skeletonof the growing rat.

MATERIAL AND METHODS

Twenty-six growing male albino rats of theSprague-Dawley strain, with a mean body weight of100 g at the beginning of the experiment, were used.They were housed 4 or 5 in each cage and were fed andwatered ad libitum. The experimental protocol wasapproved by the Ethics Committee of Göteborg Uni-versity. The animals were randomly separated into 2

equal groups (n � 13) according to diet; 1 (hard-diet

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Katsaros et al 353

group) received the ordinary diet of hard pellets (Diet R47, Astra Ewos A B, Södertalje, Sweden), and the other(soft-diet group) received the powdered form of theordinary diet mixed with water in standardized propor-tions (2:5, R 47 and water).18

The experimental period started when the ratswere 4 weeks old and lasted 42 days; all rats wereweighed 3 times a week. On days 0, 14, and 28 of theexperimental period, calcein was injected into allanimals (1.5 mg per 80 g).

After the rats were killed, their heads were taken forpreparation of undecalcified sections. The specimenswere fixed in 10% buffered formalin, dehydrated inalcohol of progressively increasing concentrations, andembedded in methylmethacrylate.19 Frontal sections120 mm thick were prepared with a Leitz-Saw mic-rotome.

To study homologous representative sections fromeach animal, the last section before the anterior palatineforamen 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 previouslyreported differences in the width of the facial suturesunder different functional demands were found mostlyin this part of the snout.17

The selected sections were viewed with an Olym-pus BHS fluorescence microscope (objective Olympus

Fig 1. a, Ventral (left) and lateral (right) view ofline denotes section taken as common basis foof rat skull (base section). IN, Internasal suture,suture.

SPLAN FL2). An excitation filter and barrier filters

(Olympus 20BP490, 17AFC, and 17O515) were usedto obtain light at wavelengths of 435-490 nm. The levelof bone apposition at the 3 times of calcein injectionwas marked with separate fluorescing lines (Fig 2).Because the lines from the first injection could not beseen in all areas because the bone had resorbed or thefluorescing lines were weak, bone apposition wasquantified from day 14 to the end of the experimentalperiod.

Optical recording was done with a Sony DXC-LS1P CCD color video camera on an Olympus U-PMTVC camera adapter. The images were loaded intoa 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 � 1125pixels and 24-bit color resolution. The images werethen checked for measurability and motion artifacts byusing Photoshop 4.0 (Adobe) and stored as uncom-pressed TIFF files for further processing. For calibra-tion purposes, a 100-�m scale was also recorded in thesame 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

ull. Vertical lines mark 3 sections used. Longertion; b, schematic illustration of frontal sectionaso-premaxillary sutures; IP, inter-premaxillary

rat skr selecNP, n

StarCalc from the StarOffice Suite V5.1a (SUN Micro-

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354 Katsaros et al

systems) on the same machine, with Microsoft ExcelData format for transportability.

First, the scale image was measured, giving aresolution of 520 pixels/mm, so that subsequent calcu-lations were performed by using 0.52 pixels/�m as aconversion factor. The internasal, naso-premaxillary,and inter-premaxillary sutures were studied. The boneapposition was measured at 5 approximately equidis-tant locations distributed over the length of each sutureperpendicular to the bone apposition sites. Measure-ments were made from the day-14 line to the day-28line, and from the day-14 line to the final bone level atthe sutural edges.

The sutural bone apposition was to be presented asthe mean of the 2 sutural edges. For that purpose, themean of all measurements was calculated for eachsuture at each section. This calculation method wasused to compensate for any differences in the way thetissue blocks were sectioned (angle between saw andlong axis of the skull).

For each rat, the mean of all 3 sections was used.

Fig 2. Undecalcified section of interpremaxillary sutureunder fluorescent microscopy. Days 0, 14, and 28:fluorescing lines marking level of bone apposition atcalcein injection. Day 42: bone surface at sutural edge.Fluorescing line at day 0 is weak and missing in someparts of suture because that bone has been resorbed.

The Student t test was used to test for differences

between the hard-diet and the soft-diet groups. Tocompensate for differences in growth, sutural boneapposition between days 14 and 42 was also comparedbetween the 2 groups after correction for final weightand nose-tail distance (at day 42) by using analysis ofcovariance (ANCOVA). Paired t tests were used forcomparison between the different sutures.

Double measurements on 20 sections were madeto evaluate the method error. The magnitude of thecombined method error was calculated according toDahlberg’s formula: Se � ��d2/2n, where d is thedifference between 2 registrations of a pair, and n is thenumber of the double registrations.20 The averagecombined method error was found to be 2.3 �m. Pairedt tests were used between the 2 series of measurementsto test for systematic errors; no statistically significantdifferences were found.

RESULTS

The rats’ mean body weights, weight gains, andnose-tail distances were similar in the 2 groups. In thehard-diet group, however, there were outliers for finalmean body weight (at day 42) and weight gain (Table I).

In both groups, greater bone apposition was foundbetween days 14 and 28 than between days 28 and 42of the experimental period (Table II).

The bone apposition rate was found to be lower inthe soft-diet group than in the hard-diet group in allsutures studied (Table II). Only in the inter-premaxil-lary suture was the difference between days 14 and 28not statistically significant. Especially in the naso-premaxillary suture, bone apposition between days 14and 42 was 26% less in the soft-diet group than in thehard-diet group. Also, after the correction for finalweight and nose-tail distance, the magnitude of thedifferences in bone apposition between the groups didnot change (Table III).

The amount of bone apposition differed betweenthe sutures in both groups. The greatest bone appositionwas registered in the nasal side of the naso-premaxil-lary suture, followed by the inter-premaxillary sutureon both sides. The internasal suture showed the leastbone apposition (Table II). The differences between thesutures were statistically significant (P � .0001).

DISCUSSION

In this study, the influence of masticatory musclefunction on sutural bone apposition was investigated byreducing the loads applied to the skull during mastica-tion. This reduction was experimentally induced byfeeding the animals a soft diet. This experimentalmodel enables the effects of an altered function to be

observed in an intact masticatory system.1

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Katsaros et al 355

Our findings suggest that reduced masticatory mus-cle function results in decreased bone apposition rate inthe plane facial sutures of the growing rat. The fact thatthe differences in bone apposition between the 2 groupswere still present after correction for final weight andnose-tail distance shows that the bone apposition ratewas not influenced by intergroup differences in overallgrowth. Thus, the differences in bone apposition be-tween the 2 groups could be attributed to the effect of

Table I. Means and standard deviations (SD) of weightdifferences

Hard-diet grou(n � 13)

Mean

Weight (g) (day 0) 118.6Weight (g) (day 42) 393.1Weight gain (g) (days 42-0) 274.5Nose-tail distance (mm) (day 42) 44.7

*1 outlier: weight � 558.0 g; weight gain � 436.0 g.NS, not significant.

Table II. Means and standard deviations (SD) of overalintergroup differences

Bone ap

Hard-diet group(n � 13)

Mean SD

Internasal (14-28) 42.6 4.1Internasal (14-42) 77.5 6.6(14-28/14-42) x % 55.0Naso-premaxillary (14-28) 88.1 9.0Naso-premaxillary (14-42) 157.5 16.4(14-28/14-42) x % 55.9Inter-premaxillary (14-28) 57.1 5.8Inter-premaxillary (14-42) 103.3 10.7(14-28/14-42) x % 55.3

(14-28/14-42) x %, Ratio of bone apposition between days 14-28 anNS, Not significant.

Table III. Differences in bone apposition between hard-d(day 42)

Withoutcorrection

Mean SE Signifi

Internasal (14-42) 13.7 2.4Naso-premaxillary (14-42) 41.2 7.4Inter-premaxillary (14-42) 10.0 4.3

SE, Standard error of mean.

the altered masticatory muscle function.

The differences in the amount of bone appositionbetween the sutures show the different modelingpattern in the various regions of the facial skeleton.In the naso-premaxillary suture, most of the boneapposition occurred at the nasal part, whereas thepremaxillary part showed weak apposition. In con-trast, cortical bone apposition is much higher in theupper part of the premaxilla than in the nasal bone.These regional differences in bone apposition are

ose-tail distance of rats and significance of intergroup

Soft-diet group(n � 13)

SignificanceMean SD

120.5 5.7 NS370.7 18.0 NS250.2 19.8 NS

44.1 1.3 NS

apposition, based on all sections, with significance of

n (�m)

Soft-diet group(n � 13)

Significance (P value)Mean SD

36.2 3.6 �.000563.8 5.4 �.000556.766.5 11.3 �.0005

116.3 21.1 �.000557.256.0 6.6 NS93.3 11.1 �.0360.0

14-42 expressed as percentage.

d soft-diet groups for final weight and nose-tail distance

After correction

(P value) Mean SE Significance (P value)

05 12.7 2.4 �.0000505 39.4 7.8 �.00005

9.2 4.5 �.05

and n

p

SD

5.761.5*61.1*

1.8

l bone

positio

d days

iet an

cance

�.000�.000�.04

important in accommodating the changes in form and

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356 Katsaros et al

spatial orientation of the nasal and premaxillarybones during growth.

The decreased bone apposition rate under reducedfunctional demands might be related to the decreasedwidth of the sutural space recorded in a previous studywith a comparable experimental design.17 The smallerseparation of the sutures under reduced functionaldemands might be the result of decreased tension in theperiosteal membrane in the suture because of lowercontractive forces of the elevator muscles. This can beexplained by the alteration in the development of theelevator muscle fiber size and composition21 as well astheir contractive capacity22 because of the prolongedalteration of masticatory function. The tension in theperiosteal membrane has been shown to regulate thedisplacement of bones in the craniofacial region.23,24

The findings of Behrents et al,25 who showed experi-mentally in living monkeys that bite forces couldprovide sufficient tensile strain to cause separation ofthe sagittal suture of the skull, support this aspect.

The decreased sutural bone apposition rate mighthave contributed to the decreased width of the premax-illa under reduced functional demands at areas underdirect muscle influence near the site of their attach-ment.16

The decrease in the contractive forces of the eleva-tor muscles after reduced masticatory muscle functionalso results in decreased occlusal loading, which caninfluence growth even in regions without muscle at-tachment. The smaller width and the lower ossificationrate of the synchondrosis of the midpalatal suture foundin rats fed a liquid diet support this aspect.26

Our findings differ from those on cranial sutures inminiature pigs when the magnitude of sutural strain wasnot correlated with sutural bone apposition rate.13

However, this either could reflect the anatomical andfunctional differences between facial and cranial su-tures or might be due to the anatomical differencesbetween the 2 species. A rat’s snout is positioned muchmore anteriorly relative to the posterior occlusion thana miniature pig’s snout, and thus the biomechanics ofthe facial skeleton during mastication are probablydifferent. Moreover, in the rat, the anterior and middleparts of the internasal suture are butt-ended, whereasthey are interdigitated in the miniature pig. That couldimply that tensile strains are applied during masticationin the rat and compressive strains in the miniaturepig.10,11,13,27

CONCLUSIONS

Our findings suggest that the bone apposition rate inthe facial sutures in the anterior facial skeleton of the

growing rat is significantly 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 Göteborg, andFrank Sick, University of Saarland, for their technicalassistance and Professor Martin van’t 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), AmericanJournal of Orthodontics and Oral Surgery (1938-1947), American Journal ofOrthodontics (1948-1986), and American Journal of Orthodontics and DentofacialOrthopedics (1986-present)

1915 to 1932 Martin Dewey1931 to 1968 H. C. Pollock1968 to 1978 B. F. Dewel1978 to 1985 Wayne G. Watson1985 to 2000 Thomas M. Graber2000 to present David L. Turpin