2.1.

Experimental Design

The study was approved by the Ethics Committee for the Use of Animals (2010/003045) of the Aracatuba Dental School, Universidade Estadual Paulista (UNESP). A total of thirty-two 50- to 60-day-old female adult rats (Rattus norvegicus albinus, Wistar) weighing approximately 200 g were used. The animals were maintained in cages and were fed with balanced chow (NUVILAB, Curitiba PR, Brazil) containing 1.4% calcium (${\mathrm{Ca}}^{++}$) and 0.8% phosphate (${\mathrm{PO}}_{4-}$). The animals received water ad libitum for 10 days during acclimatization and after the start of the experiment.

The animals were divided into four groups ($n=8$) according to the induction of osteoporosis and the drug treatment received. Twenty-four rats underwent bilateral ovariectomy and were divided into the following groups: ovariectomized rats fed a low ${\mathrm{Ca}}^{++}$ (0.1%) and ${\mathrm{PO}}_{4-}$ (0.5%) diet (RHOSTER Ind. Com., Vargem Grande Paulista, SP, Brazil) without drug treatment (OG, negative control) and ovariectomized rats fed a low ${\mathrm{Ca}}^{++}$ and P diet and treated with raloxifene (RG, test 1) or alendronate sodium (AG, test 2). Eight rats underwent a sham surgery, exposing the ovaries such that the rats underwent the same surgical stress as the other groups. The animals in this group were fed a balanced diet containing 1.4% ${\mathrm{Ca}}^{++}$ and 0.8% ${\mathrm{PO}}_{4-}$ (SG; positive control; NUVILAB, Curitiba PR, Brazil).

The chronological sequence of events in this study is detailed in Fig. 1. Before ovariectomy, the rats were subjected to estrous cycle analysis for more than three regular cycles. After ovariectomy, animals in the RG and AG groups were fed with a low ${\mathrm{Ca}}^{++}$ and ${\mathrm{PO}}_{4-}$ diet. The remaining groups were fed a balanced standard diet. On the 8th day after ovariectomy, drug treatments were initiated by gavage with $1\mathrm{mg}·{\mathrm{kg}}^{-1}·{\text{day}}^{-1}$ RG^8,28 or with $0.1\mathrm{mg}·{\mathrm{kg}}^{-1}·{\text{day}}^{-1}$ alendronate sodium (AG).²⁹

Fig. 1

Chronological flowchart of the events in this study.

On the 52nd day after ovariectomy or sham surgery, $20\mathrm{mg}/\mathrm{kg}$ calcein (Sigma Chemical Company, St. Louis, Missouri) was intramuscularly administered, and on the 80th day, $20\mathrm{mg}/\mathrm{kg}$ alizarin red (Sigma Chemical Company) was administered. Fluorochromes were diluted to 1.5 mL in deionized water with a magnetic stirrer (Max Labor, Presidente Prudente, SP, Brazil). The animals were euthanized 18 days after the application of alizarin red by overdose of the inhaled anesthetic, halothane (Tanohalo, Cristália, Cristal Pharma, Contagem, MG, Brazil).

2.2.

Laboratory Procedures

The right jaws of the rats were removed and fixed in 10% paraformaldehyde solution. After 48 h, the specimens were washed in running water for 24 h and dehydrated in increasing concentrations of alcohol. The bones were then embedded and infiltrated in a solution of acetone and methyl methacrylate slow (MMAL) (Clássico, Artigos Odontológicos Clássico, São Paulo, SP, Brazil) at a ratio of $1:1$. This was followed by three MMAL baths. Benzoyl peroxide catalyst (1%, Riedel—de Haën AG, Seelze—Hannover, Germany) was added to the last bath. The specimens were placed in glass jars covered with a lid and were maintained in an oven at 37°C for 5 days until the resin polymerized.

After polymerization, the blocks containing the specimens were reduced with a “Maxcut” drill mounted on a Kota countertop motor (Strong 210, São Paulo, SP, Brazil), parallel to the long axis of the jaw (sagittal plane). The specimens were then subjected to progressive manual wear with a polishing machine (ECOMET 250PRO/AUTOMET 250, Buehler, Lake Bluff, Illinois) with sandpaper (granulation of 120, 300, 400, 600, 800, and 1200; Carbimet 2, Buehler, Lake Bluff, Illinois) under fluorescent light, until a thickness of $80\mu \mathrm{m}$ was reached, as measured by a digital caliper (Mitutoyo, Pompeia, SP, Brazil).

The histological sections were mounted on slides with mineral oil (Petrolato Líquido, Mantecor, Taquara, RJ, Brazil) and fixed with coverslips and enamel to prevent oil leakage and section drying.

2.3.

Scanning Confocal Laser Microscopy

Longitudinal scans of the right jaw of the alveolar bone adjacent to the apical third of the maxillary central incisor were obtained by a Leica CTR 4000 CS SPE microscope (Leica Microsystems, Heidelberg, Germany), using a $\times 10$ objective ($\text{original}\text{amplification}\times 100$) (Fig. 2). The area was chosen considering the reference of the bone close to the upper right incisor. Therefore, the tooth was taken as the reference, and then the area of the bone around this tooth was evaluated in each sample.

Fig. 2

Area corresponding to the alveolar bone selected for analysis by confocal microscopy (white asterisks). White arrow indicates the region of the right upper incisor.

The images that were obtained from different sections were compressed in order to obtain the best fit. After the selection of the section thickness, all of the slices were obtained and the software performed the z-stack that allowed achieving the best fit of images that represented the section from each animal of the experimental groups. The images started from the beginning of the fluorescence thus representing, in our methodological approach, the beginning of calcification or bone formation (calcium precipitation in the collagen organic matrix).

These images had a dimension of $1\times 1{\mathrm{mm}}^2$ and corresponded to optical sections of $512\times 512\text{pixels}$. Sections ($2\mu \mathrm{m}$) were scanned over 2.5 min. Therefore, 28 sections were obtained for each $56\text{-}\mu \mathrm{m}$ slide. The barrier filters used were BP $530/30\mathrm{nm}$ and 590 LP, combined with activation of double dichroic $488/568\mathrm{nm}$, and the photomultiplier was set to 534 for calcein and 357 for alizarin. The codes 534 and 357 nm represent the filters that allow the visualization of the fluorochromes. They are located in the microscope and when the light that comes from the mercury lamp passes through these filters and reaches the sample in the slice, it is possible to visualize the colors of the fluorochromes. One of the filters enables visualization of calcein (blue filter) and the other, alizarin (green filter).

The images obtained by confocal microscopy were reconstructed through the stack of the software that is installed to manipulate the confocal microscope (Leica CTR 4000 CS SPE, Leica Microsystems, Heidelberg, Germany); the alveolar bone showed two overlapping fluorochromes (calcein and alizarin; Fig. 3). The overlap represents the superposition of calcium that was precipitated in both periods, thereby showing the conversion of old bone to new bone. These images were saved in TIFF format and transported to the ImageJ software (Processing Software and Image Analysis, Ontario, Canada). Using the “color threshold” tool, each image was standardized according to hue, saturation, and brightness to reveal the fluorochromes. First, the calcein was highlighted, and the “measure” tool was used to provide the corresponding area in $\mu {\mathrm{m}}^2$. The same procedure was performed for the alizarin, obtaining data related to the dynamics of the alveolar bone tissue.

Fig. 3

Alveolar bone images obtained using confocal microscopy. (a) Bone area stained with calcein (green); (b) bone area stained with alizarin (red); and (c) merged images of both fluorochromes.

Bone turnover, in this methodological approach, is represented by the difference between old bone (green) and new bone (red). Fluorochromes are chemical compounds that have the property of binding to calcium in the moment of the precipitation on the organic bone matrix. Therefore, the extent of fluorochrome labeling represents the quantity of calcium precipitation, thereby enabling the measurement of the bone formation event. Another aspect to be considered is the period when the fluorochromes were injected; as the first one was calcein (green), the bone filled with the green fluorochrome represents the old bone. The last fluorochrome injected was alizarin; therefore, the bone filled with the red fluorochrome represents the new bone. Considering these points, it can be said that the different colors represent the different ages of the formed bone.^30,31 Bone tissue dynamics is represented by the bone turnover that is observed through the renewal bone represented by the red fluorochrome. The greater the prevalence of the red fluorescence the greater the formation of new bone while the green fluorescence represents the old bone. The dynamics make it possible to observe the two situations in the same slice, i.e., to observe what is new and what is old.^30,31

2.4.

Statistical Analysis

Sigma Plot 12.3 (San Jose, California) software was used for statistical analysis. The Shapiro–Wilk test ($p=0.953$) was conducted to determine the homoscedasticity, i.e., whether the results were homogeneous. Data were analyzed with two-way analysis of variance (ANOVA; experimental groups: four levels; fluorochromes: two levels). Significant results were statistically evaluated with Tukey’s post-hoc test. For all tests, a $p$-value less than 5% was considered significant.