**2.10. Histomorphometric analysis**

#### *2.10.1. Fluorescent dyes used for double labeling in histomorphometric analysis*

Fluorochromes are calcium binding substances that are preferentially taken up at the site of active mineralization of bone known as the calcification front, thus labeling sites of new bone formation. They are detected using fluorescent microscopy on undecalcified sections. Labeling bones with fluorochrome markers provides a means to study the dynamics of bone formation. The rate and extent of bone deposition and resorption can be determined using double and triple fluorochrome labeling sequences. The sequential use of fluorochromes of clearly contrasting colors permits a more detailed record of events relating to calcification. Flurochromes commonly used in mammals include tetracycline, calcein green, xylenol or‐ ange, alirazin red, and hematoporphyrin. Calcein is a fluoresces bright green when com‐ bined with calcium [27].

*2.10.3. Method of analysis*

The bone around the lower second molar is centrally located within the mandibular arch, and because of the parallel alignment of the buccal and lingual roots this made a precise refer‐ ence when frontal sections are produced [29]. To conduct the histomorphometric analysis it is essential to use a digitizing morphometry system to measure bone formation indices. The system consists of a confocal laser scanning microscope (LSM510, Carl Zeiss Co. Ltd., Jena, Germany), and a morphometry program (LSM Image Browser, Carl Zeiss Co. Ltd., Jena, Germany). Bone formation indices of the periosteal surfaces of the alveolar/jaw bone in‐

the standard nomenclature described by [30]. The calcein-labeled surface (CLS, in mm) is calculated as the sum of the length of double labels (Thomas *et al.*) plus one half of the length of single labels (sL) along the entire endosteal or periosteal bone surfaces; that is, CLS ═ dL + 0.5sL [31]. The mineral apposition rate (MAR, in μ / day) is determined by dividing the mean of the width of the double labels by the interlabel time (7 days). The bone formation rate (BFR) is calculated by multiplying MAR by CLS [32]. Based on the reference line along the long axis of the buccal root, the area superior to the root apex was considered alveolar bone, while the area inferior to the root apex was considered the jaw bone. The lingual side of the bone is

**Figure 14.** Schematic drawing of observation regions for dynamic bone histomorphometry. The periosteal surfaces were delimited into 4 areas as alveolar crest (region 1), alveolar bone (region 2), buccal surface of the jaw bone (re‐

The periosteal surfaces of the mandible are divided into four regions for analysis (Fig. 14.):

The obtained results in our study showed that in the alveolar bone (region 2), there was a significant decrease in the MAR (Fig. 15A) BFR (Fig.15B) recorded in the DM group com‐ pared to the control group. However, in the alveolar crest (region 1), the MAR and the BFR in the control and the DM groups were not significantly different. (P < 0.05). In the buccal surface

gion 3), and inferior border of the jaw bone (region 4).

*2.10.4. Histomorphometric indices*

excluded, because the existence of the incisor root may influence bone formation.

/μm2

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clude mineral apposition rate (μm/day) and bone formation rate (μm3

### *2.10.2. Calcein administrations and sections preparation*

The steps needed for detecting the double labeling involves the following:


**Figure 13.** Frontal sections of the mandibular second molar area. (A) Control; (B) DM. Fluorescent labeling on the peri‐ osteal surface indicates new bone formation.

#### *2.10.3. Method of analysis*

**2.10. Histomorphometric analysis**

414 Type 1 Diabetes

bined with calcium [27].

Japan).

*2.10.2. Calcein administrations and sections preparation*

maldehyde in 0.1 M phosphate buffer (pH 7.4).

osteal surface indicates new bone formation.

*2.10.1. Fluorescent dyes used for double labeling in histomorphometric analysis*

The steps needed for detecting the double labeling involves the following:

**•** Mandibles are dissected and fixed in the same solution for 24 hours.

ces of buccal and lingual roots of the lower second molar [28].

Fluorochromes are calcium binding substances that are preferentially taken up at the site of active mineralization of bone known as the calcification front, thus labeling sites of new bone formation. They are detected using fluorescent microscopy on undecalcified sections. Labeling bones with fluorochrome markers provides a means to study the dynamics of bone formation. The rate and extent of bone deposition and resorption can be determined using double and triple fluorochrome labeling sequences. The sequential use of fluorochromes of clearly contrasting colors permits a more detailed record of events relating to calcification. Flurochromes commonly used in mammals include tetracycline, calcein green, xylenol or‐ ange, alirazin red, and hematoporphyrin. Calcein is a fluoresces bright green when com‐

**•** Rats are subcutaneously injected with 50 mg/kg body weight calcein fluorescent marker on day 21 and day 28 after STZ injection [28]. The time difference between the 2 injections is one week to be able to compare the amount of bone formed during this period (Fig. 13).

**•** Sacrifice of all animals by transcardiac perfusion under deep anesthesia using 4% parafor‐

**•** All specimens are embedded in polystyrene resin (Rigolac, Nisshin EM Co. Ltd., Tokyo,

**•** Undemineralized ground frontal sections are processed to show the crown and both api‐

**Figure 13.** Frontal sections of the mandibular second molar area. (A) Control; (B) DM. Fluorescent labeling on the peri‐

The bone around the lower second molar is centrally located within the mandibular arch, and because of the parallel alignment of the buccal and lingual roots this made a precise refer‐ ence when frontal sections are produced [29]. To conduct the histomorphometric analysis it is essential to use a digitizing morphometry system to measure bone formation indices. The system consists of a confocal laser scanning microscope (LSM510, Carl Zeiss Co. Ltd., Jena, Germany), and a morphometry program (LSM Image Browser, Carl Zeiss Co. Ltd., Jena, Germany). Bone formation indices of the periosteal surfaces of the alveolar/jaw bone in‐ clude mineral apposition rate (μm/day) and bone formation rate (μm3 /μm2 /day), according to the standard nomenclature described by [30]. The calcein-labeled surface (CLS, in mm) is calculated as the sum of the length of double labels (Thomas *et al.*) plus one half of the length of single labels (sL) along the entire endosteal or periosteal bone surfaces; that is, CLS ═ dL + 0.5sL [31]. The mineral apposition rate (MAR, in μ / day) is determined by dividing the mean of the width of the double labels by the interlabel time (7 days). The bone formation rate (BFR) is calculated by multiplying MAR by CLS [32]. Based on the reference line along the long axis of the buccal root, the area superior to the root apex was considered alveolar bone, while the area inferior to the root apex was considered the jaw bone. The lingual side of the bone is excluded, because the existence of the incisor root may influence bone formation.

**Figure 14.** Schematic drawing of observation regions for dynamic bone histomorphometry. The periosteal surfaces were delimited into 4 areas as alveolar crest (region 1), alveolar bone (region 2), buccal surface of the jaw bone (re‐ gion 3), and inferior border of the jaw bone (region 4).

The periosteal surfaces of the mandible are divided into four regions for analysis (Fig. 14.):

#### *2.10.4. Histomorphometric indices*

The obtained results in our study showed that in the alveolar bone (region 2), there was a significant decrease in the MAR (Fig. 15A) BFR (Fig.15B) recorded in the DM group com‐ pared to the control group. However, in the alveolar crest (region 1), the MAR and the BFR in the control and the DM groups were not significantly different. (P < 0.05). In the buccal surface (region 3) and inferior borders (region 4) of the jaw bone the MAR (Fig.15A) and BFR (Fig. 15B) were significantly suppressed compared with those in the control group (P < 0.05). Most of the periosteal surfaces in the mandibular regions of the control group showed significant‐ ly higher values recorded for the mineral apposition rate and the bone formation rate when compared to the DM group. These results agree with previous studies that recorded dimin‐ ished lamellar bone formation in DM rats' femur and may suggest an association between the DM condition and the decreased number and function of osteoblasts [16, 19]. The alveolar crest region was the only region that did not show a significant difference in the mineral apposition rate and the bone formation rate parameters among the two groups; this may be attributed to the unique nature of this region exhibiting a highly intensive bone remodeling process especially during the teeth eruption that decreases toward the base of the socket [33], however further studies are needed to elaborate the detailed pattern of bone growth at the alveolar crest region.

*2.10.5. Microtomography of the mandible (Micro-CT)*

ence East Corporation, Tokyo, Japan)

stage.

size of 15 μm / pixel.

Tokyo, Japan).

ed by morphing.

Micro-computed tomography (micro-CT) has rapidly become a standard technique for the visualization and quantification of the 3D structure of trabecular bone. Bone architecture and mineralization are generally considered to be important components of bone quality,

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In our study all specimens were imaged by micro-CT (inspeXio SMX-90CT; Shimadzu Sci‐

**•** After removing only the soft tissue, the mandibular plane is set orthogonal to the sample

**•** Three dimensional images of each hemi mandible are acquired with a resolution voxel

**•** Raw data are obtained by rotating the sample stage 360 degrees. Then, slice images are prepared using multi-tomographic image reconstruction software (MultiBP; Imagescript,

**•** The resulting gray-scale images are segmented using a low-pass filter to remove noise

**•** The volume of interest (VOI) is drawn on a slice-based method starting from the first slice containing the crown of the first molar and moving dorsally 100 slices [34, 35], in the area of the alveolar crest (Between the buccal and lingual roots of the second molar at the cer‐ vical region); and the buccal surface of the jaw bone [29]. Trabecular bone was carefully contoured on the first and the last slice, while the intermediate slices were first interpolat‐

**•** For observation and analysis of reconstructed 3D images, 3D trabecular structure analysis software (TRI/3D-BON; RATOC System Engineering, Tokyo, Japan) is used [36]. Recon‐ structed 3D images were prepared from slice images using the volume rendering method,

**•** The following parameters are measured: tissue volume (TV), bone volume (BV), bone sur‐

**•** Four properties of the trabeculae are evaluated: trabecular thickness (Tb.Th), trabecular

face (BS), bone surface / bone volume (BS/BV), bone-volume fraction (BV/TV).

number (Tb.N), trabecular separation (Tb.Sp), and Trabecular space (Tb.S) [36].

and determine bone strength in conjunction with bone mineral density.

*2.10.6. Protocol adopted to examine the mandible using Micro-CT*

and a fixed threshold to extract the mineralized bone phase.

to analyze the microstructure of the bone (Fig. 16).

**Figure 15.** A) The changes in mineral apposition rate (MAR) of the mandible between the control group and the DM group. Alveolar crest (region 1, upper 1/2 of the tooth root, near the tooth crown). Alveolar bone (region 2, lower 1/2 of the tooth root, near the root apex). Buccal surface of the jaw bone (region 3). Inferior border of the jaw bone (re‐ gion 4). The data are expressed as means ± SD. N = 5 for each group. \*Significantly different from controls, with (p<0.05). (B) The changes in the bone formation rate (BFR/BS) of the mandible between the control group and the DM group. Alveolar crest (region 1, upper 1/2 of the tooth root, near the tooth crown). Alveolar bone (region 2, lower 1/2 of the tooth root, near the root apex). Buccal surface of the jaw bone (region 3). Inferior border of the jaw bone (region 4). The data are expressed as means ± S.D. N = 5 for each group. \*Significantly different from controls, with (p<0.05).
