Bone micro-architectural analysis of mandible and tibia in ovariectomised rats

Animals and administration procedure

All animal experiments were approved by the Animal Care and Use Committee of Peking University Health Science Center (Approval number: LA2014233, Beijing, China). Female Sprague Dawley rats aged eight weeks (n = 15) were purchased and a bilateral OVX or sham operation was performed using standard methods. Four weeks after the OVX or sham operation, rats were assigned randomly into three groups of five: sham rats injected with vehicle (sham group); OVX rats injected with vehicle (OVX group); and OVX rats injected with zoledronate (100 μg/kg) subcutaneously at days 1 and 8 (OVX-ZOL group). Three months later, the rats were killed and the mandible and the tibia from each animal were thoroughly dissected free from soft tissue and fixed in 10% neutral-buffered formalin, and used for micro-CT and undecalcified histological sections.

Micro-CT analysis

The mandibles and tibias were studied using a compact fan-beam-type tomography system (Inveon MM CT, Siemens AG, Munich, Germany). In brief, the mandible or tibia was located and scanned in its entirety, being rotated 360° in 360 equiangular steps.¹³ Images were scanned at a resolution of 8.82 μm, tube voltage of 80 kV, tube current of 500 μA and exposure time of 1500 ms. The images consisted of 1536 slices with a voxel size of 17.64 μm in all three axes. Two-dimensional images were used to generate 3D reconstructions using multimodal 3D visualisation software (Inveon Research Workplace, Siemens). In order to evaluate the influence of scanning resolution on the results from micro-CT analysis, the same specimen was scanned at resolutions of 8.82 μm, 13.57 μm, 35.32 μm, 43.74 μm and 104.84μm, with all other parameters being kept the same.

For the mandible, the region of interest (ROI) was obtained from all trabecular bone under the first molar domains (Fig. 1) as previously described.¹⁷ For the tibia, the ROI was drawn 2 mm from the nadir of the proximal epiphysis. An appropriate threshold was adjusted exactly to the mineralised bone phase. The threshold levels distinguishing trabecular bone from bone marrow for mandible and tibia in sham, OVX and OVX-ZOL groups are shown in Table I. The parameters were calculated as follows: bone volume density (BV/TV), bone surface density (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp).²³

Fig. 1

This representative image from a micro-CT shows that the region of interest (ROI) on the mandible in the axial plane and on the tibia in the coronal plane (the yellow outline represents the ROI on mandible and tibia). Scale bar = 1 mm.

Histological sectioning and staining

The mandible and tibia were fixed and dehydrated in ethanol followed by embedding in destabilised methyl methacrylate resin under vacuum at room temperature. Next, the sections were ground and polished to 20 μm to 30 μm by the EXAKT precision cutting and grinding system (EXAKT Apparatebau GmbH & Co KG, Norderstedt, Germany) and stained with haematoxylin and eosin (H&E).¹³ A similar ROI, as previously defined in the micro-CT measurement, was analysed by means of a semi-automatic histomorphometric analysis system (Bio-Quant Inc., San Diego, California). The undecalcified histological slices stained by H&E were automatically photographed by Nikon inverted microscope and the images were transferred to graphic workstation. Next, the ROI of the undecalcified histological slices was drawn using the BQ Imaging Toolkit (Bio-Quant Inc., San Diego, California). After the threshold value was set, the histomorphometric parameters were automatically calculated by BIOQUANT OSTEO Image Analysis Systems (Bio-Quant Inc., San Diego, California). Of note, trabecular diameter (Tb.Dm) in two-dimensional histology is instead of the Tb. Th from 3D histomorphometric parameters of micro-CT.

Statistical analysis

Quantitative data for correlations of the histomorphometric parameters on mandible or tibia between micro-CT and histological sections are presented as bivariate linear regressions using SPSS 16.0.1 software (IBM, Armonk, New York). The coefficient of variation (CV) was calculated as the standard deviation (sd) percentage of the mean. Coefficients of correlation (r) and determination (r²) are given for the goodness of fit. Additionally, one-way analysis of variance (ANOVA) was undertaken for comparison within groups; p < 0.05 was considered statically significant. Quantitative data of the histomorphometric parameters are presented as mean and sd.

Stereology-based comparison of histological and corresponding micro-tomographical sections of mandible and tibia

Compared with the images of histological sections in the first row of Figure 2, the 3D reconstruction images (second row) showed that there were still a slight reduction of bone mass in trabecular boundaries, which might be correlated with the loss of low-density tissue induced by radiograph in micro-CT, such as new bone and osteoid (Fig. 2).

Fig.

These images show the bone micro-architecture of the a) mandible and b) tibia among sham, ovariectomised (OVX) and zoledronate-injected ovariectomised (OVX-ZOL) groups in the coronal plane. The first row represents a single histological section. The second row represents a 3D reconstruction corresponding to histological section. Scale bar = 1 mm.

The evaluation of the accuracy of micro-CT analysis of mandibles and tibias at different scanning resolutions

The radiographs of mandibles and tibias from the same rat in the sham group became less clear as the scanning resolution decreased. The boundary between trabecular bone and bone marrow of tibias and mandibles could not be clearly distinguished at resolutions below 8.82 μm and 13.57 μm, respectively (Fig. 3). There were unpredictable changes in the results of morphometric parameters at different resolutions; the data obtained at the highest resolution were similar to those obtained by histological analysis. It was therefore concluded that the most appropriate resolution at which to scan was 8.82 μm.

Fig. 3

Radiographs of the mandible (top) and tibia (bottom) from the same rat at all five scanning resolutions. The radiographs became less clear as the scanning resolution decreased. Scale bar = 1 mm.

Histomorphometric parameters of mandible correlations between histological indices and micro-CT

The results indicated that the mean calculated by micro-CT was slightly overestimated in BV/TV, BS/BV and Tb.N compared with that of histology in the remaining three groups. The Tb.Sp average was calculated to be roughly equal between the two methods. According to the formula (Tb.Dm = 4/(BS/BV), Tb.Th = 2/(BS/BV)), Tb.Dm was equal to twofold Tb.Th in number (Tb.Dm = 2* Tb.Th).²⁴ Hence, the mean of Tb.Th from micro-CT was equal to Tb.Dm by histology, and the two parameters are combined hereinafter to simplify the analysis. The coefficients of variations (CV) of histomorphometric parameters from micro-CT were lower when compared with their corresponding histological CVs.

Our results demonstrated that there were significant correlations in the mandible between histological indices and micro-CT analysis for all five histomorphometric parameters (Fig. 4). Moreover, for the mandible, good correlation was found between the two methods for BV/TV, Tb.Th, and Tb.Sp, with the mean percentage differences being no more than 5%. Larger differences, although still relatively small, were found for BS/BV and Tb.N.

Fig.

Histological indices and micro-CT analysis correlations from the same histomorphometric parameters of the mandible; a) bone volume density; b) bone surface density; c) Tb.Dm (Tb. Th); d) trabecular number; e) trabecular separation. A total of 15 specimens among sham, ovariectomised, and zoledronate-injected ovariectomised groups were used for correlations. All regressions were highly significant.

The correlations of the histomorphometric parameters of the tibia between histological indices and micro-CT

Our results demonstrated that the 3D values from micro-CT were overestimated in the BV/TV compared with 2D measurements obtained from histological sections. The averages of the other parameters were inconsistent between the two methods. However, the CV of histomorphometric parameters from micro-CT was lower compared with their corresponding histological ones. Unexpectedly, the CVs of the tibia were higher compared with those of the mandible, especially in the OVX-ZOL group. In the OVX-ZOL group, the CV value of the tibia was dramatically higher than that of either sham or OVX groups. This was associated with the pyknotic bone trabecular plate induced by zoledronate below the proximal epiphysis (Fig. 2b).

In addition, for the tibia, there were significant correlations between histological indices and micro-CT analysis for all five histomorphometric parameters (Fig. 5). However, for the tibia, good correlation between the two methods was only found in Tb.Th. The mean percentage differences of the other four parameters in the tibia were higher than those of the mandible.

Fig.

Histological indices and micro-CT analysis correlations from the same histomorphometric parameters of the tibia; a) bone volume density; b) bone surface density; c) Tb.Dm (Tb. Th); d) trabecular number; e) trabecular separation A total of 15 specimens among sham, ovariectomised and zoledronate-injected ovariectomised groups were used for correlations. All regressions were highly significant.

Histomorphometric parameter correlations between the mandible and tibia by histological indices and micro-CT analysis

There were significant correlations observed in BV/TV, BS/BV, and Tb.Sp between mandible and tibia, respectively (Fig. 6). Consistent with the correlation of histological analysis, the results of micro-CT showed that the more significant correlations were found in BV/TV, BS/BV, Tb.N, and Tb.Sp between mandible and tibia (Fig. 7). These data have confirmed that the variation of histomorphometric parameters in the mandible was associated with that of the tibia in OVX-induced osteoporotic or zoledronate-treated rat models.

Fig. 6

Correlations from the same histomorphometric parameters between the mandible and tibia by histological indices analysis; a) bone volume density; b) bone surface density; c) trabecular separation. A total of 15 specimens among sham, ovariectomised, and zoledronate-injected ovariectomised groups were used for correlations. All regressions were highly significant.

Fig.

Correlations from the same histomorphometric parameters between the mandible and tibia by micro-CT analysis; a) bone volume density; b) bone surface density; c) trabecular number; d) trabecular separation. A total of 15 specimens among sham, ovariectomised, and zoledronate-injected ovariectomised groups were used for correlations. All regressions were highly significant.

Comparison of histomorphometric parameters among sham, OVX, and OVX-ZOL groups by undecalcified histological sections and micro-CT

For undecalcified histological sections of the tibia, the OVX group significantly decreased in BV/TV, Tb.Dm, and Tb.N compared with sham and OVX-ZOL groups. Moreover, there were significant increases in BS/BV and Tb.Sp in the OVX group compared with the sham and OVX-ZOL groups. The OVX-ZOL group dramatically increased in BV/TV and decreased in BS/BV compared with the sham group. Consistent with undecalcified histological sections, micro-CT analysis of the tibia showed that the values of BV/TV, Tb.Th, and Tb.N in the OVX group were lower than in sham and OVX-ZOL groups with the exception of the Tb.N of the OVX-ZOL group. Moreover, there were significant increases in BS/BV and Tb.Sp of the OVX group compared with sham and OVX-ZOL groups. However, the parameters of BV/TV and Tb.Th in the OVX-ZOL group were higher than that of the sham group. Meanwhile, the BS/BV in the OVX-ZOL group was lower than the sham group.

In undecalcified histological sections of the mandible, there were significant decreases in BV/TV and Tb.Dm of the OVX group compared with sham and OVX-ZOL groups. Moreover, the OVX group showed a dramatic increase in Tb.N and Tb.Sp compared with the sham group. The OVX-ZOL group showed a significant increase in Tb.N compared with the sham group. Meanwhile, in the OVX-ZOL group, Tb.Sp was dramatically decreased compared with the sham and OVX groups. Consistent with undecalcified histological sections, the micro-CT results of the mandible showed that the OVX group significantly decreased in BV/TV and Tb.Th and increased in BS/BV compared with the sham and OVX-ZOL groups. Of note, inconsistent with the tibia, the Tb.N of the mandible in the OVX group exhibited a significant increase compared with the sham group using both methods, which might be associated with new bone lacunae of the alveolar septum induced by bone resorption in the mandible (Fig. 2a). The OVX-ZOL group showed a significant decrease in Tb.Sp compared with the OVX group. These aforementioned data further corroborate the finding that undecalcified histological sections and micro-CT have an excellent ability to evaluate OVX-induced osteoporosis and osteoporotic treatment. Moreover, consistent with the tibia, bone histomorphometry on the mandible also revealed the presence of osteoporosis or effect of osteoporotic treatment in an ovariectomised rat model.