The effect of ovariectomy and 2 antiresorptive therapeutic agents on bone response in rats: A 3-dimensional imaging analysis

Objective. The aim of this study was to evaluate bone mineral density (BMD) and microarchitecture in femurs and maxillary bones of ovariectomized (OVX) rats treated or not treated with alendronate (ALD) or odanacatib (ODN).

Study Design. Twenty rats were divided into groups: SHAM, OVX, OVX/ALD, and OVX/ODN. After 12 weeks, the femurs and maxillae were removed and subjected to 3-dimensional analysis by micro–computed tomography. Results were analyzed with 1-way analysis of variance and Tukey’s post hoc test (α = 0.05).
Results. OVX decreased maxillary and femoral BMD and altered femoral microarchitecture (P < .05). The drugs increased BMD of both types of bones, but only ALD maintained the phenotype similar to the SHAM group. The action of ALD was limited to the femoral trabecular separation (Tb.Sp). OVX and the drugs had no effect on the microarchitecture of the maxilla (P > .05). Conclusions. ALD and ODN therapy increased BMD in both bones after ovariectomy. ALD was more successful than ODN in preserving the morphology of bone similar to the SHAM group. ALD maintained the phenotype for Tb.Sp in the femur, but ODN did not. In the maxillae, neither ovariectomy nor the 2 antiresorptive drugs had significant effects on microarchitecture. (Oral Surg Oral Med Oral Pathol Oral Radiol 2018;■■:■■–■■)

Osteoporosis is a metabolic bone disease characterized by decrease of bone mineral density (BMD), leading to skeletal fragility and increased risk of fractures.1,2 Studies evaluating osteoporosis and the therapeutic agents for the prevention and treatment of this disease often use ovari- ectomized rats as an experimental model, approved by the U.S. Food and Drug Administration, to mimic post- menopausal conditions, such as reduced BMD.3 Micro– computed tomography (micro-CT) is considered the gold standard to evaluate bone morphology and microarchitecture in rodents and other animal models, presenting excellent reproducibility and accuracy.4,5

The effect of osteoporosis on BMD and the microarchitecture of maxillary bones has been studied, but the results are still controversial.6,7 There have been reports of a decrease in BMD after ovariectomy (OVX) in rats, with differences of results depending on the type of bone. Long bones and vertebrae are more sensitive than maxillary bones to the estrogen deficiency that occurs after OVX.8 Some studies have shown that maxillary bones are minimally affected by OVX,8-10 whereas some others have demonstrated changes in alveolar bone po- rosity, with a resulting decrease in BMD in the mandibles of ovariectomized rats.7,11 In addition, there are large dis- crepancies among the studies using ovariectomized rats8 with regard to animal age at the time of surgery, type of bone, and duration of the experimental period.

The prevention and treatment of bone loss and osteo- porosis after menopause is of utmost importance in maintaining bone integrity and preventing possible fractures.12 Bisphosphonates are the first-choice drugs for the pharmacologic treatment of osteoporosis.13 Alendronate (ALD), a bisphosphonate containing nitro- gen, is considered a drug capable of preventing bone loss induced by estrogen deficiency12,14 and is associated with increased BMD and reduced risk of fracture.15 However, an association between the systemic use of alendronate and a high incidence of osteonecrosis of the jaw has been suggested.16 Therefore, there is an urgent need to develop new potential alternative antiresorptive agents,17 with the mechanism of action based on the inhibition of bone resorption.

Odanacatib (ODN) is an antiresorptive drug that acts specifically on the inhibition of cathepsin K, an enzyme secreted by osteoclasts that degrades type I collagen. ODN has been shown to increase BMD of the spine and hips,18 as well as the radius and tibia,19 and has the ability to reduce bone resorption without decreasing the number of osteoclasts and preserving bone formation.20 ODN leaves the osteoclasts alive and unaffected but inhibits bone resorption by inhibiting cathepsin K activity.21 The mechanisms by which cathepsin K inhibition increases bone modeling, mainly at the periosteal surface, remain unclear.22 Currently, ODN is being clinically tested (phase III) in the treatment of postmenopausal osteoporosis. Al- though cathepsin K inhibitors have the ability to suppress ovariectomy-induced bone loss in rats,23 there are no studies evaluating the effect of ODN on maxillary bones. Therefore, the objective of the present study was to eval- uate BMD and microarchitecture in femurs and maxillary bones of ovariectomized (estrogen-deficient) rats, sub- jected or not subjected to treatment with bisphosphonate (alendronate) or a cathepsin K inhibitor (odanacatib) for prevention of osteoporosis.

MATERIAL AND METHODS

Animals

After approval of the research project by the Institu- tional Animal Research Ethics Committee (process number #2013.1.1404.58.4), 20 female Wistar rats (Rattus norvegicus albinus), approximately 12 weeks old, were selected from the University’s animal facility and kept under standard laboratory conditions. The rats were fed standard laboratory animal chow and were offered fil- tered water ad libitum.

The animals were randomly assigned to 4 groups: (1) SHAM: animals subjected to sham surgery, (2) OVX: animals subjected to OVX, (3) OVX/ALD: animals sub- jected to OVX and treatment with ALD, and (4) OVX/ ODN: animals subjected to OVX and treatment with ODN.

The rats were anesthetized by an intramuscular injec- tion of 10% ketamine (55 mg/kg weight) and 2% xylazine (10 mg/kg), and the ovaries were excised,
as a whole, bilaterally in the experimental groups. In the SHAM control group, surgery was simulated, and the ovaries were gently moved and immediately returned to their origi- nal position, only to simulate surgical stress, as described by Orrico et al.24 The success of OVX was confirmed by monitoring the animal’s weight and the weight of the uterus at the day of euthanasia, considering that OVX is associated with weight gain and uterine atrophy.14

Administration of drugs

Administration of the study drugs started 1 day after OVX. The OVX/ALD group received 2 doses per week (1 mg/ kg) of chemically pure sodium alendronate (Alendronate Monosodium Trihydrate; Galena Química e Farmacêutica Ltda., Campinas, Brazil), diluted in distilled water and administered by inserting a gavage needle into the oral cavity, totaling 24 doses during the experimental period of 12 weeks.14 The OVX/ODN group received, by gavage, a total of 7 doses (2.12 mg/kg) of ODN (Santa Cruz Bio- technology Inc., Santa Cruz, CA) diluted in polyethylene glycol (PEG 400) (2.5 mg/mL) every 12 days, totaling also 12 weeks. The dose and frequency of administra- tion were calculated by interspecies extrapolation by allometric rules from mice to rats, based on previous data presented by Hao et al.25

For administration of the drugs via gavage, the animals were fasted for 2 hours. A curved stainless steel oral gavage needle specific for rats (Ciencor Scientific Ltda., São Paulo, SP, Brazil) with a cannula measuring 1.2 mm in diameter, 38 mm long, and 2.25 mm ball tip, was used to inject the solution directly into the gastrointestinal tract, as described by Nelson-Filho et al.26 After gavage, the animals were maintained for a few minutes in an upright position to avoid the occurrence of gastroesophageal reflux and/or irritation and to ensure complete ingestion of the volume administered in the procedure. The animals were weighed weekly to adjust the concentration of the ALD and ODN solutions according to the changes occurring in weight over time.

After the experimental period, the animals were eu- thanized by using ketamine hydrochloride (Ketamina Agener, União Química Farmaêutica Nacional S/A, Embu-Guaçu, Brazil) for anesthesia, xylazine hydro- chloride (Dopaser; Caleir S.A., Barcelona, Spain), and carbon dioxide inhalation. The bones were surgically removed with the aid of sterilized scissors and a scalpel blade and stored in 70% ethyl alcohol for fixation until scanning by micro-CT.

Micro-CT to analyze BMD and bone microarchitecture

A high-resolution micro-CT device (SkyScan 1174 v2; Bruker-microCT, Kontich, Belgium) was used to quan- tify the BMD and 3-dimensional (3-D) microarchitecture parameters in the femur and maxillae. Cross-sectional images of each specimen were reconstructed with spe- cific software (NRecon version 1.6.3; Bruker-microCT), with ring artifact correction of value 10.

The regions of interest (ROI) were limited to the cortical-free trabecular bone, which is more delicate and comparable with osteoporotic human bone.27 In each bone, the ROIs were selected according to Liu et al.8: the tra- becular alveolar bone in the interradicular septum of the maxillary first molar and the axial trabecular alveolar bone located 3 mm under the growth plate.

For accurate calculation of BMD, appropriate cali- bration of the CTAn software (v. 1.14.4.1 + , Bruker- microCT, Kontich, Belgium) was performed by using calcium hydroxyapatite phantoms of known density (0.25 and 0.75 g/cm3). Once the BMDs of the phantoms were calibrated in the CTAn software, the following structur- al parameters of the ROI were calculated: BMD; bone volume fraction (bone volume/total volume = BV/TV), defined as the fraction of bone in the total ROI; trabecu- lar thickness (Tb.Th); trabecular number (Tb.N), defined as the number of times that a trabecular is crossed by others per unit of length; and trabecular separation (Tb.Sp).7,8

An experienced examiner, who was blinded to the groups, was previously trained and calibrated to perform all analyses. Data were expressed as the means ± stan- dard deviations. All statistical analyses were carried out with Graph Pad Prism 4.0 software (Graph Pad Soft- ware Inc., San Diego, CA). Data were subjected to 1-way analysis of variance (ANOVA) and Tukey’s post hoc anal- ysis. With respect to uterine weight and body weight, data were subjected to the Student’s t-test. The level of sta- tistical significance was 5%.

RESULTS

Uterine weights

The success of OVX was confirmed by the animals’ uterine weights. There was significant uterine atrophy in the OVX animals (P < .0001) at the day of euthanasia, with 73% reduction in uterine weight compared with that of the SHAM group. There was also a significant dif- ference between the uterine weights of the SHAM and OVX animals (P < .001). The SHAM group’s uterine weight increased by an average of 22%, whereas in the OVX group, the uterine weight increased by 34%.

BMD and bone microarchitecture of the femurs The results of femoral BMD are presented in Figure 1. The BMD means of groups SHAM, OVX, OVX + ALD, and OVX + ODN were, respectively, 0.35 ± 0.03, 0.21 ± 0.01, 0.30 ± 0.04, and 0.26 ± 0.03 g/cm3. There was a statistically significant difference (P < .001) between the SHAM and OVX groups, which means that the ovari- ectomized animals presented lower BMD compared with the SHAM animals. The administration of ALD and ODN increased femoral BMD, but only ALD maintained the phenotype similar to the SHAM group, considering that there was no statistical difference between the OVX/ ALD and SHAM groups (P > .05).

The results of femoral bone microarchitecture are pre- sented in Figure 2. There were statistically significant differences between the SHAM and OVX groups (P < .05) with respect to BV/TV, Tb.N, and Tb.Sp, which means that OVX caused alterations in these param- eters. The drug ALD acted in the femoral Tb.Sp maintaining the phenotype, considering that there was no statistical difference between the OVX/ALD and SHAM groups. Both ALD and ODN decreased BV/TV and Tb.N, but neither was able to maintain the same phe- notype of SHAM animals. With regard to Tb.Th, no statistically significant difference could be found between the groups (P > .05).

BMD and bone microarchitecture of the maxillae The results of maxillary BMD are presented in Figure 3. BMD means of the SHAM, OVX, OVX + ALD, and OVX + ODN groups were, respectively, 1.11 ± 0.02, 0.97 ± 0.08, 1.09 ± 0.03, and 1.02 ± 0.04 g/cm3. There was a statistically significant difference (P = .004) between the SHAM and OVX groups; that is, ovariectomized animals presented lower BMD compared with the SHAM animals. The administration of ALD and ODN in- creased the maxillary BMD, but only ALD maintained the phenotype similar to the SHAM group, considering that there was no statistical difference between the OVX/ ALD and SHAM groups (P > .05).

The results of maxillary bone microarchitecture are pre- sented in Figure 4. There were no statistically significant differences (P > .05) between the groups with respect to all evaluated parameters (BV/TV, Tb.Th, Tb.N, and Tb.Sp). Figures 5 and 6 illustrate specimens representative of the different groups.

Discussion

Numerous indicators can be used to describe trabecular bone microarchitecture and cortical bone morphology. According to Bouxsein et al.,4 at least 4 parameters (BV/ TV, Tb.Th, Tb.Sp, and Tb.N) should be used to evaluate trabecular bone microarchitecture; for this reason, they were selected for evaluation of both types of bones in the present study.

In laboratory studies, micro-CT is considered the gold standard for evaluation of the mineral density and microarchitecture of trabecular bone,4 being a high- resolution, nondestructive technique that produces a 3-D image of the bone structure.28 The effect of estrogen deficiency, evaluated by micro- CT, depends on the type of bone evaluated; long bones (e.g., femur and tibia) and vertebral bones are the most commonly used in research studies. The findings of the present study demonstrated that the reduction in estro- gen levels, 12 weeks after OVX, affected the BMD and microarchitecture of the long bone (femur). In agree- ment with our results, Hao et al.29 and Liu et al.8 reported alterations similar to those of the present study, after evalu- ating the trabecular BMD and microarchitecture in the femurs of ovariectomized rodents, 12 weeks after OVX. Using micro-CT, Cline-Smith et al.30 also observed a de- crease in BMD and BV/TV in the tibiae of ovariectomized mice compared with SHAM animals.

In dentistry, the effect of reduced estrogen levels must be further explored. Changes in the subchondral bone of the temporomandibular joint, including reduction of BV/ TV, Tb.Th, and Tb.N, and increase of Tb.Sp were observed in ovariectomized rats 10 weeks after OVX, compared with SHAM animals.31 Additionally, ovariectomized rats presented alterations in BMD and bone microarchitecture in the peri-implant region,32 which led other authors to suggest that in women with osteoporosis, dental implant surgery should be carried out with caution.28,33 In addi- tion, a more accentuated progression of periodontal disease in ovariectomized animals, with greater verti- cal bone loss compared with SHAM animals, has been demonstrated.34

Although the effect of osteoporosis on the mineral density and microarchitecture of maxillary bones has been investigated, the results are inconsistent.35 OVX causes a significant reduction in the BMD of the femur and tibia, but it does not have the same effect on maxillary bones.9 In the present study, a decrease of maxillary BMD was observed in the ovariectomized rats, in contrast to the find- ings of Ishihara et al.9 but in agreement with Dai et al.7 and Liu et al.,8 who also observed lower maxillary BMD in rats 12 and 24 weeks after OVX, respectively. In con- trast, in the present study, OVX did not cause statistical changes in maxillary bone microarchitecture 12 weeks after surgery, whereas Dai et al.7 observed such changes, possibly because of the use of animals younger than ours (6 weeks of age). The results of the present study are in agreement with those of previous investigations that re- ported long bones responding faster to OVX compared with maxillary bones.8,35 We hypothesized that this oc- curred because healthy long bones have lower mineral density compared with maxillary bones (0.35 ± 0.03 and 1.11 ± 0.02, respectively, in the present study).

Additionally, studies evaluating the impact of OVX on mandibular alveolar bone showed alterations in bone microarchitecture and greater porosity when compar- ing ovariectomized and SHAM animals.8,11,28,29,36,37 In general, the longer the elapsed time after OVX, the more severe are the alterations in alveolar bone. Con- sidering that the greatest alterations in alveolar bone have been shown to occur within 12 weeks after OVX,35,38 this post-OVX time was used in the present study. Like- wise, the age of the rats at the time of OVX surgery determines the maturity of their skeleton, and it is known that changes in the bone structure of an adult rat are closer to those observed in the mature human skeleton. Thus, the rat must be at least 3 months old at the time of surgery,38 which was the case in the present study.

However, little is known about the effect of antiresorptive drugs on maxillary bones. Some studies have evaluated the effect of bisphosphonates in dentist- ry. Jee et al.39 reported that ALD aids in the healing of postextraction sockets and in reducing adjacent alveo- lar bone loss in estrogen-deficient rats. Abtahi et al.40 demonstrated that local treatment with bisphosphonate improves dental implant fixation. Anbinder et al.41 found that OVX reduces alveolar BMD, and, in agreement with our results, they observed that ALD was efficient in treat- ing this condition and restoring BMD to the same levels of SHAM-animals. However, those authors evaluated al- veolar BMD from an analysis of radiographs. To date, there are no studies using micro-CT to specifically eval- uate BMD and bone microarchitecture of interradicular maxillary bone in ovariectomized animals under treat- ment with ALD, and this makes our findings not comparable with the current literature.

It has been shown that ODN can inhibit the develop- ment of apical periodontitis, bone resorption, and immune response in mice.25 In addition, this drug reduces exter- nal root resorption induced by orthodontic treatment42 and inhibits the innate immune response in periodontitis.43 In the present study, ODN increased maxillary BMD in the ovariectomized animals, but it was not able to main- tain the phenotype similar to the SHAM group. The lack of similar studies evaluating the effect of ODN on max- illary bones hinders a reliable comparison with other studies.

Therefore, further research should be performed to es- tablish knowledge of the effect of both drugs—ALD and ODN—on maxillary bones. The present study demon- strated that OVX resulted in maxillary alterations and ALD was effective in maintaining the BMD phenotype similar to that of the SHAM group. This knowledge is critical for the dental surgeon for the correct manage- ment of patients with osteoporosis.

CONCLUSIONS

Reduction of estrogen levels induced by OVX was able to affect femoral and maxillary BMD and femoral microarchitecture. The 3-D changes caused by estro- gen deficiency were greater in long bone than in maxillary bone. Treatment with ALD or ODN increased the BMD of both types of bones evaluated, but only ALD was capable of maintaining the phenotypical morphology similar to that of the SHAM group. Additionally, ALD maintained the phenotype for trabecular separation in the femur, but ODN did not. In the maxillae, neither ovari- ectomy nor the 2 antiresorptive drug had significant effects on microarchitecture.