INTRODUCTION

According to the Ministry of Health, Labour and Welfare, Japanese calcium intake has been insufficient.1 It can decrease bone mineral content (BMC) and peak bone mass (PBM). The PBM is reached at the end of puberty or at the beginning of adulthood in humans, and is one of the most important parameters to predict individual possibility of fracture.2 Thus, for recommending a necessary means of prevention of fracture, an accurate evaluation of PBM is very useful.3–5 In addition, in daily clinical practice when we think of alveolar bone treatment with implants, BMC is one of the very important parameters in making prognosis or postoperative evaluation. BMC is an essential parameter for the prevention, treatment and maintenance of periodontitis. Though there are comparisons of BMC among various bone types under the condition of insufficient calcium intake in adulthood, those in the growing period are rarely reported.

Once BMC is reduced, we have to recover it in order to enjoy a healthy life. In general, bone grafting and some medicines to increase the BMC are the major means of recovering BMC and bone volume.6–9 In these treatments, evaluations of BMC is a valuable means for confirming bone reconstruction. BMC is generally evaluated by translating the opacity of the bone into that of an equivalent thickness of aluminum wedge, which is called microdensitometry.10–12

Insufficient calcium intake can affect maxillofacial growth, too.13 Maxillofacial growth is often evaluated by cephalometric analysis. So, evaluation of BMC and cephalometric analysis are important means of evaluating the effect of insufficient calcium intake on skeletal development.

In previous evaluations of BMC using digital radiographs such as Dixel,11 we could use only small imaging plates. Moreover, in cephalometric analysis, researchers conducting basic studies evaluated a great number of samples and manual analysis required much time. In the present study, we used a VISTA SCAN (Dürr Dental GmbH & Co. KG, Bietigheim-Bissingen, Germany) that was able to analyze larger animal bones and broader human regions (57 × 76 mm), and created a new computer program to analyze the results. Further, in order to save time and decrease workload, we created software for semi-automatic cephalometric analysis, too. Using those systems, we investigated the effects of a low calcium diet on BMC of lower alveolar bones, femurs and tibias, and on maxillofacial growth in growing male Wistar rats.

MATERIALS AND METHODS

Bone Mineral Content

We created computer programs for evaluation of BMC and to perform cephalometric analysis using Visual Basic 6.0 (Microsoft Co, Seattle, U.S.A.) and Image Kit 7 (Microsoft Co, Seattle, U.S.A.). The digital radiographs consisted of 256 gradations, from 0 to 255. They were used as parameters of BMC when we evaluated BMC by microdensitometry. Microdensitometry is the means of evaluating BMC by translating it into the thickness of aluminum wedge equivalent which was taken in the radiograph together with the bone sample. By comparing mean gradations of pixels in the region of interest to the gradations of aluminum wedge equivalent, we can get BMC as a value of aluminum thickness. It is one of the major means of BMC evaluation.1112

First, one of the radiographs was displayed on the screen. The scanning area on the aluminum wedge was fixed by a right-click. The area ranged from the ordinate of the right-click ±5 pixels (the area between green lines in Figure 1). After scanning, the correlation coefficient was calculated.

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Second, both ends of the bone were fixed by left-clicks (red lines in Figure 1), after which the scanning began with the third left-click at any point above the bone. The scanning area ranged the mid-point of the fixed bone ends ±17 pixels (the blue area in Figure 1). In previous studies, a densitometer (PDS-15; KONICA MINOLTA HOLDINGS, INC, Tokyo, Japan) was used in evaluating BMC. However, that device has a slit size of 10 × 500 μm, which is too narrow to determine whether the scanning area is proper or singular. The scanning area ranged enough in our present study.

Cephalometry

A radiograph of the lateral view was displayed on the screen. Two directions of the coordinate axes were applied with the upper-left corner defined as the origin, and the downward direction along the Y axis and rightward direction along the X axis considered to be positive. There were 27 fixed points and 29 measurement items for length14 (Table 1 and Figure 2).

Table 1.  Landmarks and Measurement Items Used for Cephalo metric Analysis

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Second, we plotted all the points except for U1′, U2′, L1′, L2′, and Co′, specifying each with the option button. The 22 points were stored in the computer and displayed on the screen at the same time (Figure 3). In this manner, we were able to obtain most of the length using a formula to get a distance between 2 points.

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In order to obtain the distances of U1-U1′, U2-U2′, L1-L1′, L2-L2′, and Co-Co′, the computer program calculated formulae of lines A-N and Pg-Gn, and then used a formula to obtain the distance between a point and a line. Thus, we could obtain 5 measurement items, and could accomplish all of the measurements for our cephalometric analysis.

RESULTS

All rats were weighed at the beginning of the experiment, the day that the diet of the LCSD group was changed to standard (at 4 weeks), and on the last day of the experiment (at 6 weeks). No significant differences were observed in increments of weight among the groups, as shown in Table 2.

Table 2.  Comparison of Weight Increments in First 4 Weeks and Following 2 Weeks of the Experiment (Unit: g)

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For confirmation of the reproducibility of the results obtained with our computer software, we evaluated values according to the F-test by a one-way ANOVA as shown in Table 3, and we confirmed the reproducibility of our newly created computer programs.

Table 3.  F-values for Confirmation of Reliability on our Software

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For evaluation of BMC, we found significant differences; Co > LC (P < .01), Co > LCSD (P < .01), and LCSD > LC (P < .05) in the lower alveolar bones, Co > LC (P < .01), Co > LCSD (P < .01), and LCSD > LC (P < .01) in femurs, and Co > LC (P < .01) and LCSD > LC (P < .01) in the tibias (Table 4).

Table 4.  Significant Differences in Comparisons of Bone Mineral Content of Alveolar Bones, Femurs, and Tibias (Unit: mmAl)

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In cephalometric analysis, no significant differences were observed among the groups for the Total Skull and Neurocranium parameters. On the other hand, significant differences were observed in some of the other parameters; Co > LCSD (P < .05) in Mu-Pr, Co > LCSD (P < .05) and LC > LCSD (P < .05) in Iu-Pr, Co > LCSD (P < .05) in U1-U1′, and Co > LCSD (P < .01) in U2-U2′, which reflect size of Maxilla. Co > LCSD (P < .01) and Co > LC (P < .01) in Co-Go, Co > LCSD (P < .05) and Co > LC (P < .01) in Co-Gn, Co > LC (P < .01) in L1-L1′, and Co > LC (P < .01) in L2-L2′, which reflect growth in the superior-inferior direction of the mandible, as were Co > LCSD (P < .05) and LC > LCSD (P < .05) in Il-Bl, LC > Co (P < .01) in Pg-Gn, and LCSD > LC (P < 0.05) in Il-Id, which show growth in the anterior-posterior direction of mandible (Table 5).

Table 5.  Significant Differences in Changes in Linear Measurements of Craniofacial Skeletons (Unit: mm)

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DISCUSSION

It is generally known that BMC decreases due to a low calcium diet,15–18 which was confirmed by our experimental results. BMC in the Co group was significantly higher than that in the LC group in the lower alveolar bones, femurs, and tibias. Previous studies have also reported the effects of a low calcium diet on various kinds of bones.1920

Nevertheless, it was interesting that the recovery of BMC differed according to bone kind after changing from a low calcium diet to a standard one (Table 4). In the lower alveolar bones, recovery of BMC was moderate after changing from a low calcium diet to a standard one. On the other hand, in tibias, the BMC in the Co and LCSD groups was significantly higher than that in the LC group, while a significant difference was not observed between the Co and LCSD groups. Thus the BMC recovered completely because of changing from the low calcium diet into a standard one. In the femurs, recovery of BMC by changing from the low calcium diet to a standard one was greater than in the mandibles. Those results indicated that BMC in the mandibles and femurs recovered to some extent, though not completely, by changing from a low calcium diet to a standard one.

In the normal bone growth process, bone is deposited when there is sufficient calcium intake, however, when insufficient the bone remodeling process resorbs existing bone in order to maintain linear bone growth and periosteal expansion.2122 Further, an excess secretion of PTH due to secondary hyperparathyroidism might accelerate bone resorption in the present LC and LCSD groups. After changing from a low calcium diet to a standard one, we observed significant differences in the recovery of BMC according to the type of bone. In general, the lower alveolar bone is more sensitive to PTH than other bones,23 which was considered to be the reason why BMC in the lower alveolar bones did not recover well by changing from the low calcium diet to a standard one. Also, the lower alveolar bones are thought to have a relatively high turnover and such active bones tend to show bone loss, due to a small imbalance between bone resorption and formation.23 We concluded that once BMC in the lower alveolar bones decrease, it rarely recovers completely, thus it is very important to prevent a reduction of BMC, especially in alveolar bones.

Our cephalometric analyses showed that the growth of mandibles in the superior-inferior direction was decreased significantly in the rats that consumed a calcium-restricted diet. It is known that bone formation and calcification are inhibited by a low calcium diet that continues for a long period. This is because resorption of pre-existing bone occurs in order to meet the calcium needs of longitudinal bone growth and periosteal expansion, resulting in reductions in bone size and weight.24 Moreover, insufficient calcium intake could induce reduction of growth hormone, which resulted in reductions in bone size.25 This was observed in the mandibles, which have greater growth potential.26 On the other hand, we are not able to provide a satisfactory explanation as to why the growth of the mandible in an anterior-posterior direction was significantly increased in the rats that consumed the calcium-restricted diet. Values of LCSD in Iu-Pr, U1-U1′, U2-U2′, Il-Bl, and L2-L2′ were significantly lower than those of Co or LC. These parameters can be influenced by tooth contour, so the significant differences could be caused by constitutional tooth contour, habit, and other factors rather than calcium-restricted diet.

Though we allocated the groups randomly, the bone samples are small and we cannot know if they were initially the same. So, there may be a limitation in applying these data to smaller bone samples. Also, our software was made for 2 dimensional evaluations, but we hope to adapt it for 3 dimensional evaluations in the future.

We created new computer programs to evaluate BMC and to perform cephalometric analysis in our present study. Many researchers and general practitioners will be able to evaluate BMC with larger images with minimum exposure. In addition, much time can be saved and the workload reduced while performing cephalometric analyses. They can be revised on demand according to individual needs. Because of their usefulness, we hope that our computer programs will be utilized widely by many researchers and dental practitioners, and play an important role in basic studies, health promotion and treatment.

CONCLUSIONS

• Preventing BMC reduction in the lower alveolar bone is important and our computer programs can play an important role in detection.

• Development of the mandible in an anterior-posterior direction was accelerated following consumption of a low calcium diet, whereas that in the superior-inferior direction was inhibited.