radiographic measurements of alveolar bone loss in the rat

Archs oral Bid Vol. 27. pp. 651 to 658. 1982 0003-9969/82/0X0651-08$03.00/O

Printed in Great Britain. All rights reserved Copyright 0 1982 Pergamon Press Ltd

RADIOGRAPHIC MEASUREMENTS OF ALVEOLAR BONE LOSS

IN THE RAT

RUTH GAEGAUF-ZOLLINGER, J. J. BURCKHARDT* and B. GUGGENHEIM

Department of Oral Microbiology and General Immunology, Dental Institute, University of Zurich, Plattenstrasse 11, CH-8028 Zurich and

*F. Hoffmann-La Roche & Co. AG, CH-4002 Basel. Switzerland

Summary-A new method to evaluate alveolar bone loss in rodents is described. The palatal and lingual halves of maxillae and mandibles were radiographed. On enlarged positive prints, 5 vertical distances were drawn at defined sites from the cemento-enamel junction to points revealing fully intact bone structure. These were either located on the alveolar crest or at the depth of intrabony defects. These distances were recorded with a trace-reading pen coupled to a computer. Results were expressed in mm for each site separately and totals (left plus right values) for either maxillae or mandibles were calculated. This technique was compared to other methods for evaluating alveolar bone loss, using the jaws of rats subjected to a gnotobiotic regime in which the degree of bone loss was low. It was demonstrated that the measurement of vertical distances based on radiography by which also intrabony defects were defined was accurate, reproducible and more sensitive than other means of evaluating bone loss.

INTRODUCTION

Animal model systems are a useful tool in research concerned with the nature and aetiology of periodontal disease. As discussed by Jordan (1971), the suit- ability of a given animal model to replace man may be characterized by the terms fidelity and discrimination (Russell and Burch, 1959). Fidelity refers to the overall proportional differences and discrimination describes the extent to which one particular property may be reproduced in an animal model. With regard to the overall tissue reaction as well as to the nature and sequence of infiltrating inflammatory cells, exper- imentally-induced periodontitis in gnotobiotic rats shows sufficient discrimination (Guggenheim and Schroeder, 1974), while the degree of fidelity is better than might be expected. Rodent model systems have been used for three main research categories:

(i) To discover the periodontopathic potential of bacteria (Keyes and Gold, 1955; Jordan and Keyes, 1964; Socransky, Hubersak and Propas, 1970; Irving it u/., 1975; Crawford, Taubman and Smith, 1978a; Johnson et (I/., 1978).

(ii) To study different stages of periodontal disease, in terms of histopathological features on a light microscopic or ultrastructural level (Gupta and Shaw, 1956a; Thilander, 1961; Baer and Fitzgerald, 1966; Mulvihill et ul., 1967; Jordan, Keyes and Bellack, 1972; Guggenheim and Schroeder, 1974; Irving et a/., 1975; Miller and Ripley, 1975; Garant, 1976; Amstadt-Jossi and Schroeder, 1978; Ryder, 1980).

(iii) To study alterations of the host response during development of the disease (Guggenheim and Schroeder, 1974; Crawford, Taubman and Smith, 1978b; Burckhardt, Gaegauf-Zollinger and Guggen- heim, 1981a; Burckhardt et al., 1981b; Guggenheim et al., 1981).

Both the estimation of the periodontopathic potential of certain bacterial strains and evaluation of the

host response require the assessment of pathological changes in the periodontal tissues of experimental animals. Due to the small size of rodents and sometimes their gnotobiotic isolation in cages, the usual clinical measurements applied in man or larger animals to record inflammation and loss of attachment are impracticable. Reliable data can only be secured by autopsy. These consist of measurements of bone loss by various techniques (Costich, 1955; Keyes and Gold, 1955; Gupta and Shaw, 1956b; Guggenheim and Schroeder, 1974) or morphometric analysis on randomly-chosen tissue sections (Guggenheim and Schroeder, 1974; Amstad-Jossi and Schroeder, 1978). Morphometric analysis is too laborious to be the method of choice.

The above measurements of bone loss are based on the increments of either area or distance between the cemento-enamel junction (CEJ) and alveolar bone crest (ABC) and therefore reflect horizontal bone loss only. These methods are quite accurate and suffice in experiments in which there is much bone loss. Intra- alveolar bone craters hidden by the more elevated facial and oral ABC could not be assessed by these measurements. Such lesions, located predominantly at interdental sites, are the first defects to be observed and remain most prominent as the disease progresses (Guggenheim and Schroeder, 1974; Irving et al., 1975).

Crawford et al. (1978a) introduced a new technique for evaluating the vertical component of bone loss. A silicon-base impression material was injected into the defects. The trimmed impressions were then carefully removed and weighed. For several reasons, we found this method not convincing. Based on radiography we developed a new technique to measure vertical bone loss. The advantages of this procedure over horizontal bone loss measurements were compared using rat mandibles and maxillae from a gnotobiotic experi- ment in which bone loss was moderate.

651

652 Ruth Gaegauf-Zollinger, J. J. Burckhardt and B. Guggenheim

MATERIALS AND METHODS

Animals

The Roche Institut fur Biologisch-Medizinische Forschung AC, Ftillinsdorf, Switzerland, kept the germ-free nucleus of inbred RIC-Sprague-Dawley rats and sent us 8 parent animals from the same litter before mating. At 22 days of age (range 2G24 days), 43 rats from 4 litters were weaned and transferred to screen-bottom, stainless-steel cages, without bedding, in two plastic isolators.

To discriminate between litters, the rats were skin- marked with picric acid. Initially, 2-3 animals were caged together. Diet 2OOOS, sterilized by gamma- radiation (Eidgen. Forschungsanstalt, Wldenswil. Switzerland) and autoclaved fluoride-free tap water was available rrrl lihitum.

Orcll impluntafion of’ Actinomyces viscosus NY I

Actinomyces viscosus Ny 1 was obtained from Dr J. S. van der Hoeven, University of Nijmegen, The Netherlands. Overnight cultures, grown in actinomyces broth (BBL), were used to implant the organism orally in rats of one isolator, with two doses of 0.1 ml per animal at 27 and 28 days of age. Bacterio- logical controls were carried out as described by Gug- genheim and Schroeder (1974). The rats in the second isolator remained germ-free during the entire experimental period.

Prepurution of juns for radiography

At 26,46, 60 and 88 days of age, animals from each isolator were removed and weighed. After decapita- tion, maxillae and mandibles were removed, stained with erythrosine for plaque-scoring and rinsed with tap-water overnight. A colony of Dermesres maculutus beetles defleshed the jaws, which were individually packed into baskets made with meshed wire. After 2-3 days, the jaws were rinsed with 95 per cent etha- nol, immersed into 25 per cent ammonium hydroxide for 2 days, dried, and bleached with 30 per cent H202 for 668 h. To improve the visibility of the CEJ on radiographs. we immersed the jaws in saturated AgN03 solution for 30 min. Subsequently maxillae and mandibles were hemi-sectioned vertically (K&rig, Marthaler and Miihlemann, 1958) in mesio-distal direction, resulting in buccal halves and a palatal or lingual half, respectively. If loose molars were likely to be lost during sectioning, the jaws were embedded in KERR (Type 1. Sybron Corp., Romulus, Mich.).

Radiograph)

We taped the palatal or lingual halves, with the sectioned surface facing the film, the Scotch tape on radiographic film packages containing Kodak Industrex A film (CAT 501 3644, Kodak, Rochester, N.Y.). The preparations were radiographed using an ultra-crania T radiation machine (Isotopan, Rome, Italy) set at 30 kV, 200 mA and 2 s with a standardized distance of 70cm from the focus to the film plane. A standard consisting of a stainless-steel plate, in which two holes, 1 .OO cm apart, were drilled, was included in each series of radiographs and was used at a later stage to calibrate the computer planimeter. The radiographic films were developed for 8.5 min in Percentol (Kodak) at 24’C and manuallv minted on

Ilfospeed black and white paper of a suitable grade (Ilford, U.K.) with a 6.4-fold magnification. The buc-

cal halves of the jaws were not used for radiography because the ascending ~‘~mtus of the mandible partly covered the structures of interest.

The areas of each tooth between ABC and CEJ were marked with Indian ink with a 0.25 mm drawing pen on the prints (Fig. 1). Lines A and B were then drawn to indicate the mesio-distal distances at the CEJ and halfway between CEJ and ABC respectively. Lines vertical to the lines A were then drawn at 5 selected locations (see below).

Bone loss was evaluated by 3 measurements.

(i) By measuring the area between ABC and the CEJ of individual molars; this method is essen- tially a modification of a procedure described by Costich (1955).

(ii) By the mean vertical distance between CEJ and ABC. This distance was calculated by dividing the areas of indivjidual molars between CEJ and ABC by the mesio-distal tooth width measured either at the level of the CEJ (Fig. I. A) or halfway between CEJ and ABC (9). The resulting values in mm for the corresponding left and right molars in mandibles for maxillae

were added. A similar method was described

by Guggenheim and Schroeder (1974)

,

2mm ,

.H /_.---

_A*- .A __-_

Fig. 1. Drawings made from prints of radiographs; maxtllary teeth above, mandibular below. The zones from the alveolar bone crest (ABC) to the cementoPenamel junction (CEJ) are hatched. On the mandibular teeth, lmes A are drawn at the level of CEJ and lines B halfway between CEJ and ABC. On the maxtllary molars. the 5 lines vertical to the lines A are drawn where the distances between CEJ and

the deepest points of bone resorption were measured.

Measurements of alveolar bone loss 653

(iii) By measuring the vertical distances between CEJ and the deepest point of bone resorption at 5 defined locations, This point could be located at the level of the ABC; more often, however, translucent zones were indicative of an intrabony defect. These distances were measured (Fig. I) at the distal furcation of the first molar (1) and the furcation of the second (3) and third molar (5) perpendicularly to A. Interdentally. between the first and second (2) and the second and third molar (4) the distance was measured perpendicularly to a line drawn between the CEJ of the two adjacent molars to the deepest point of the interdental bony crater (Fig. 1).

These areas and distances were measured using a curser and a digitizer (Hewlett-Packard. Fort Collins, Cola.) connected to a computer (Commodore Busi- ness Machines, Santa Clara. Calif.). In young animals in which the third molar had not or was not fully erupted, the distances (4) and (5) were not measured.

The significances of group differences were calculated using Student’s r-test. The data obtained from one animal with either of the three methods were ordinated with one degree of freedom, the animal and not a particular tooth being the smallest experimental unit.

RESULTS

Areu of bone loss in mono-associated ruts

The mean values & SD of the areas defined by the CEJ and the ABC of the first and second molars in maxillae and mandibles are summarized in Table 1. Throughout the experimental period, the areas measured on the first molars were usually larger in mandibles than in maxillae. We observed, however, no consistent differences when we measured the areas of the second molars. These areas increased with age in both germ-free and mono-associated rats. Statisti- cally significant differences between germ-free controls and monoassociated rats, representing areas of bone loss, were present for both maxillary and mandibular molars. Sometimes this bone loss was statisti-

tally not significant because the individual values varied too much (e.g. day 60, maxillary molars),

Culculution qf mean vertical distimces

Mean vertical distances between ABC and CEJ or bone level according to Guggenheim and Schroeder (1974) were calculated by dividing the areas by the respective mesiodistal distances measured either at the CEJ (A in Fig. 1) or halfway between the CEJ and ABC (B in Fig. 1). As expected, the results showed that mono-association had no effect on the length of the mesio-distal distances. Changes with increasing age of the rats were minimal. For the sake of brevity, we will not present these data.

Figures 2 and 3 show the values obtained by dividing areas (Table 1) by distance A and distance B respectively. Between days 26 and 46, the mean distances increased by 0.4 mm in mandibular molars (panel A in Figs 2 and 3) and by about 0.2X1.3 mm in maxillary molars (panel B in Figs 2 and 3). After 46 days of age, the rate of increase diminished first in germ-free controls and later in mono-associated rats. Figures 2 and 3 clearly show that calculation of mean vertical distances by dividing the areas. delineated by ABC and CEJ. either by distance A or B gives similar results. Some variations are revealed by the levels of significance. However, neither way of calculation resulted in a consistently higher level of significance. Thus conversion of areas into mean vertical distances reveals no additional information.

Vertic,ct/ hone loss including interdental sites

Measuring the areas of bone loss and calculating mean vertical distances has the disadvantage that they neglect the crater-like intra-alveolar molar bone defects. To overcome this inaccuracy, we measured the 5 vertical distances on radiographs, including the interdental spaces (Fig. 1). The totals of both distances l-3 and 1-5 were calculated. The mean values f SD are summarized in Tables 2 and 3 for the maxillary and mandibular molars, respectively. In both groups of rats, all distances measured increased with age. The mean values from germ-free and monoassociated rats were little different at day 46, showed considerable

Table 1. Areas of bone loss in germ-free and mono-associated rats

Age in

days Oral

infection

Mandibles First molars Second molars

(mm2) (mm2)

26 none

46 none A. viscosus$

60 none A. viscosus

88 none A. viscosus

2.25 k 0.46t 0.72 k 0.35

3.40 * 0.38 1.23 k 0.15 3.42 f 0.31 1.27 k 0.12

3.54 f 0.36 1.19 f 0.22 4.29 k 0.23 *(8) 1.55 + 0.23 *(8)

4.30 * 0.34 1.29 + 0.15 4.75 * 0.41 1.60 & 0.21 **(J2)

Maxillae First molars Second molars

(mm2) (mm’)

1.63 k 0.44 0.64 i: 0.19

2.14 _t 0.21 *(8)

1.11 + 0.17 2.57 & 0.34 1.26 k 0.12

2.43 k 0.36 1.17 k 0.16 3.17 k 0.72 1.42 + 0.34

2.88 k 0.45 3.37 k 0.32 *(12)

t Means k SD. $ Overnight cultures of A. viscosus Ny 1 were used for mono-association on days 27 and 28. Statistically significant differences between germ-free and mono-associated rats are indicated as follows: *,

0.01 < p < 0.05; ** 0.001 < p < 0.01; figures in brackets = degrees of freedom. )


mm

LO- A

SECOND MOLARS SECOND MOLARS

0.2 tlrl L (1 IL 1 20 40 60 (10

DAIS OF AGE

Fig. 2. Mean vertical distances in mandibular molars (panel A) and maxillary molars (panel B) of germ-free controls (open symbols) or rats mono-associated with A. viswsus Ny I (closed symbols) as calculated by dividing the areas (Table 1) by the respective mesio-distal distances measured halfway between CEJ and ABC. Circles, first molars: triangles. second molars. The arrow indicates the time of oral infection. Mean values + SE are shown; SE are not shown if smaller than the symbol used.

Statistically significant differences are as shown in Table 1.

differences at day 60, and were clearly distinct at day

88. These differences between germ-free and mono- associated rats demonstrate the amount of vertical bone loss. It is noteworthy that vertical distances measured in the interdental space between second and third mandibular molars ceased to increase between day 60 and 88. In germ-free animals, they even de- creased during this period in the interdental space between first and second mandibular molars (Table 3). This demonstrates the age-dependent balance between the rate of tooth eruption and concomitant apposition of bone at the alveolar crest.

1.6 -

1.4 -

1.2 -

1.0 -

0.8 -

0.6 -

0.4 -

DAYS OF AGE

Rrproducihilit~ qf the meu.surrments

We tested the accuracy of the results by measuring I2 areas and 20 vertical distances of 4 rat molars on 5 separate days with at least a 7-day interval between readings. The mean values and variation coefficients of the five readings of each area or distance were calculated and the mean variation coefficients of areas and distances were determined. Both persons measured areas and vertical distances with a mean variation coefficient of less than 5 per cent. The range from 0.7 to 11.2 per cent indicates that some areas

1 1.6

1.4

SECOND MOLARS

ti 0.2 1, ’ ’ ’ ’ 1 1 20 40 60 80

DAYS OF AGE

Fig. 3. Mean vertical distances in mandibular molars (panel A) and maxillary molars (panel B) of germ-free controls (open symbols) or rats mono-associated with A. viscosus Ny 1 (closed symbols), calculated by dividing the areas (Table 1) by the respective mesio-distal distances measured at the CEJ. Circles, first molars; triangles, second molars. The arrow indicates the time of oral infection. Mean

values T SE are shown,

+I t-1 +I fl +I +I

*o-Z0-v o-d*‘3

dddddd +I fl +I 4” +I +I

Measurements of alveolar bone loss

\no drno- o-~o-y dd 6660

s +I +I +I +I +I +I m* ~~owf-4 vlw wr-m* dd dddo

t= +I +I fl +I +I fl

+I +I fl +I +I +I +I


and distances were much easier to measure accurately than others. Considering that the accuracy of the inte- grator used was limited to 8 pm, which is 2 per cent of the smallest distance. the reproducibility of the measurements was considered to be satisfactory.

DISCUSSIOY

In the past, alveolar bone resorption in rodents has been evaluated by the original methods described by Costich (1955) and Keyes and Gold (1955) or modifi- cations thereof. Costich described a system of evaluation which uses the weight of cellulose-acetate pat- terns cut from tracings of the relationship of the CEJ to the ABC. The results obtained in mg could be related to mm’ of bone loss and were an estimate of the horizontal bone loss per quadrant, from the first to the third molar. This method is quite accurate for scoring heavy bone loss. The lack of discrimination between different sites, the expression of bone loss in mg or mm’ and the inability to measure intra-alveolar bone pockets, are major disadvantages however. A modification (Guggenheim and Schroeder, 1974) using a point-counting procedure, with a coherent double-lattice test system, on standardized Kodac- rome diapositives covering the first and second molars, was certainly an improvement, The areas between CEJ and ABC were calculated separately for both molars and the resulting areas were divided by the respective mesio-distal width. resulting in the average distances between CEJ and ABC (bone level). However. intra-alveolar bone defects escaped detection

By using the cusps as reference points Keyes and Gold (1955) performed direct measurements of the distance between CEJ and ABC along the oral (i.e. palatal and lingual) aspect of all molars. Although this technique allows discrimination between different sites and the data are directly expressed in mm, it does not allow the estimation of intra-alveolar defects. Both the Costich and Keyes and Gold methods served well for measurements of the horizontal bone loss and provided comparable data (Guggenheim, 1980).

When the host response is the main object of study during the development of periodontal diseases, immune and clinical status of the host have to be evaluated. While very sensitive in the techniques are applied to follow the host response, assessment of the pathological changes by the above-mentioned bone- loss measurements are rather crude. Descriptive his- tology may reveal significant information on the pre- dominant alterations in the inflamed tissue. but unless quantitated, any association with the immune response must remain speculative. The evaluations of a representative segment of histological sections by morphometric and stereological analyses are much too laborious to be applied routinely.

Crawford er al. (1978a) published a new method for assessing bone loss in rodents which seemed to pro- vide a solution to this problem. The intrabony defects (vertical bone loss) were filled with a silicon-base impression material. which was trimmed flush with the alveolar plates, carefully removed and weighed. Due to the known density of the material, the defects could be expressed in mm’. In our hands, this method was

not satisfactory; it was impossible to distinguish between lesions at different sites, the reproducibility was poor and the sensitivity of the method was not better than measuring horizontal bone loss. This is reinforced by the authors’ figures. In contrast. the radiographic method for evaluating vertical distances described here proved to be more sensitive than the two elaborate methods for measuring horizontal bone loss. By the age of 60 days (46 days after infection), significant differences in the distances between mono- associated and germ-free rats were evident in both jaws. These distances were related to the distal furcations of the first and the furcations of the second molars and the approximal space between these teeth. These data were obtained although bone loss in the mono-associated group of rats in the present experi- ment was. for reasons which will not be discussed, much smaller than in similar studies (Burckhardt et rll., 198lb). The reproducibility of vertical bone loss measurements was satisfactory with a mean variation coefficient below 5 per cent and was only slightly higher than the coefficients calculated for horizontal bone loss estimations by measuring areas.

The mesio-distal distances measured either at the CEJ or halfway between CEJ and ABC revealed only minute changes during the experimental period and showed no consistent differences between germ-free and infected animals. It follows that the divisions of areas through the mesio-distal distance, measured at either level, resulted in most comparable data. It is therefore impossible to indicate preferentially one of these denominators for the calculation of mean vertical distances. In addition. the calculations of mean vertical distances have no advantage over the expression of horizontal bone loss for each molar in area units. This seems, however, irrelevant because the direct measurement of vertical distances is more sensitive and thus preferable to the measurement of either areas or mean vertical distances.

As was to be also expected, the direct measurement of distances on prints of radiographs has certain limi- tations. Some of these are technical and apply directly to our method. Others are related to the postnatal development and expression of periodontal structures in rodent species and are therefore not associated with any particular technique for measuring bone loss.

We realize that the main advantage of our method is to offer early and precise detection of bone loss and this is expensive in labour, patience and a number of instruments which include a suitable radiation machine, an internal-rim rotary cutting machine, a well-equipped laboratory for the development of radiographs and photographs and, not least, access to a computer coupled with a system allowing plani- metric measurements. Some skill is needed to hemi- section the jaw quadrant. Occasionally the cementoPenamel junction cannot be identified on the print. Consulting the original radiograph of such preparations solves this problem.

As shown by Hefti er al. (1980) the use of der- mested beetles for cleaning bone is not without danger. The speed of cleaning is a function of the population composition and size, amount of food offered, temperature and exposure to light. If jaws are not regularly inspected, bone may be destroyed by

Measurements of alveolar bone loss 657

these insects. As the radiographic method is not dependent on rigorously cleaned jaws, stripping off adhering soft tissue with the aid of a scalpel after prefixing in phosphate-buffered formalin, is rec- ommended.

Our measurements of vertical distances expressed in mm do not distinguish between sites with true intrabony defects (vertical bone loss) and sites on the ABC (horizontal bone loss). We therefore feel that neither the term horizontal bone loss nor the term vertical bone loss, as used in human studies, are ad- equate, because we measured in each quadrant at defined sites to the deepest point, either the alveolar crest or an intrabony defect. Crawford et a/. (1978a) pointed out that horizontal and vertical bone loss are related parameters and that the pattern of bone loss is mainly dependent on the particular organism used for infection and the length of the period in which the rats were exposed to this agent. Our findings corrob- orate these points. Intrabony defects were observed in rats monoassociated with A. z~isco.sus (Burckhardt et al., 1981a,b) and in animals infected with Gram-negative anaerobes (data not presented here). This type of lesion was preferentially observed in approximal areas and to a lesser extent in the furcations of the molars. Intra-alveolar bone defects were not observed in germ-free controls. However, it has been consistently reported that the distance between CEJ and ABC in- creases during the life span of conventional rats (Hoff- mann and Schour, 1940; Sicher and Weinmann, 1944) and in conventional mice (Gilmore and Glickmann, 1959). as well as in germ-free rats (Amstad-Jossi and Schroeder, 1978; Crawford et ul., 1978a). This process occurs preferentially on lingual and palatal aspects of the molars and has been interpreted by Belting et a/. (1953) and Amstad-Jossi and Schroeder (1978) to be a normal physiological change. These authors stated

that the molars of mice and rats continue to erupt and the alveolar bone continues to grow in height throughout the greater part of life. Furthermore, the post-eruptive, age- and growth-dependent, continuous molar eruption is in an occluso-buccal direction, while at the same time all molars drift distally. The distance between CEJ and ABC enlarges because the rate of eruption is always faster than the simultaneous rate of bone apposition at the alveolar crest, When correctly accounted for, these normal developmental changes of the rodent dentition and associated periodontal structures do in no way disqualify rodents as experimental animals for periodontal research. The physiologically normal increase in the distance between CEJ and ABC in germ-free rats varied within narrow limits when results of different experiments were compared. This is best documented by the fol- lowing data: individual sums of the distances l-3 and IL5 from mandibles originating from germ-free animals, with an average age of 97 k 6 days, which were obtained in 4 experiments within the last 3 years, were subjected to an analysis of variance. No significant differences were found between the experiments. For sites l-3 a mean distance (*SD) of 2.48 (kO.31) mm (n = 20) was calculated. while the respective mean for sites l-5 was 3.78 (_tO.41).

The precise and reproducible method for measuring vertical distances as presented here will allow certain gnotobiotic experiments without germ-free controls, a

shorter duration of experimentation and a high degree of discrimination in localizing bone loss. These advantages amply justify this technically laborious method of measuring alveolar bone loss in rodents.

Aciinowiert~emmtsWe are indebted to Dr F. Pasler for his help in radiology and we thank Mr T. Reich for setting up the computer program.

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