Osteoporosis Affects Functional

Recent studies suggest that osteoporosis, in addition to the damage caused in long bones, may cause deterioration in the jaws, especially in alveolar bone sites, with effects in the progress of periodontal disease as well as in bone healing. The aim of this study was to evaluate the effect of osteoporosis in the metabolism of rat alveolar bone osteoblasts. There were used 10 female rats divided in two experimental groups (Sham and OVX), which were ovariectomized and after 8 weeks euthanized to collect mandibular bone samples in order to isolate osteoblastic cells. The cells were cultured in 24-well plates to perform the in vitro experiments. After 7, 10 and 14 days, there were evaluated cell proliferation by MTT assay, in situ detection of alkaline phosphatase (ALP) as well as mineralized nodules and expression of genes associated to bone remodeling. Results showed that at 7, 10 and 14 days cell proliferation was lower for OVX group. In situ detection of ALP was higher at 7 days and lower at 10 and 14 days in OVX group. At 17 and 21 days, OVX group had a significative decrease of mineralization nodules. There was a downregulation in the expression of Alp , Bglap and Runx2 genes and an upregulation of Opg in OVX group, whereas Opn and Rankl modulation was similar between the evaluated groups. Our results suggest that osteoporosis has a deleterious effect on alveolar bone cells from ovariectomized rats, which might affect the treatment of diseases associated to the jaw bones.


Introduction
Osteoporosis is a systemic skeletal disease characterized by an imbalance between osteoclast bone resorption and bone formation by osteoblasts, with a consequent decrease in bone mass and an alteration of its microarchitecture, which leads to increased fragility and risk of fracture (1). There is a high vulnerability of elderly women to osteoporosis due to menopause transition, when there is a reduction of estrogen hormone, resulting in an accelerated bone resorption that exceeds its formation (2). This disease is considered a world public health problem with an increasing prevalence (3), since it is estimated that 1 in 5 men and 1 in 3 women will experience bone fractures within their lifetime (4). Excessive reactive oxygen species and inflammatory responses which occur in osteoporosis have been shown to stimulate osteoclast differentiation as well as induce osteoblast apoptosis and suppress its proliferation and differentiation (5).
Recent studies suggest that osteoporosis may cause deterioration in mandible bones besides long bones (6)(7)(8). Estrogen deficiency may promote bone mineral density (BMD) reduction and contribute to the imbalance of RANK-RANKL-OPG system, which also affects alveolar bone, increasing serum inflammatory mediators (IL-1, IL-6, and TNF), leading to bone loss (1,3) and affecting other processes such as alveolar healing after tooth extraction (9).
Although the relationship between osteoporosis and long bones is well established in the literature, its effects on mandible bone osteoblasts are not well known. Thus, the aim of this study was to evaluate the effect of osteoporosis on alveolar bone osteoblasts from ovariectomized rats, evaluating cell viability, detection and quantification of alkaline phosphatase and mineralized nodules, as well as the expression of genes associated with osteogenesis. The hypothesis is that osteoporosis condition might influence osteoblast functional activity, which could lead to alterations in mandible bone alveolar matrix formation and in its metabolism.

Material and Methods
The experimental protocol was approved by the Ethics Committee for Animal Experimentation, University of São Paulo (approval number No. 2016.1.594.58.7). Ten Wistar female rats weighting approximately 300 g were submitted to bilateral ovariectomy to induce osteoporosis (n=5) or sham operated to serve as control (n=5). Sample size calculus was a priori performed considering α=0.05, power of 95% for Rankl gene expression data for PTFE membrane (1.002±0.0713) and P(VDF-TrFE)BT membrane (0.796±0.0197) from Scalize et al. (10) investigation. The minimal sample size obtained was 6, being 3 for each group. In the present study, we selected n=5 for each group to compensate eventual losses. Sample size was calculated with software G* Power 3.0.10 (Franz Faul, Kiel University, Kiel, Germany). The animals were selected from the Central Vivarium of the University and were kept in a polyethylene box. The temperature was maintained between 23 and 24 °C with a 12-h cycle of light per day. Throughout the experiment, the animals received a selected solid diet and water ad libitum.

Ovariectomy and Sham Surgery
The rats were weighted and anesthetized by an intramuscular injection of xylazine (10 mg/kg) and ketamine (75 mg/kg) (Agibrands do Brasil LTDA, Campinas, SP, Brazil). After trichotomy and antisepsis, the ovaries were excised. Suture was performed with silk thread 4.0 (Ethicon, Johnson & Johnson, São José dos Campos, SP, Brazil). Each animal received an intramuscular injection of 0.1mL/100g body weight of veterinary pentabiotic (Pentabiotic Veterinário Pequeno Porte-Fort Dodge®, Campinas, SP, Brazil) followed by 0.2 mL/100 g of injectable Banamine® (Injetável Pet-Schering-Plough, Cotia, SP, Brazil). The success of ovariectomy was evaluated by the atrophy of uterine horns. Euthanasia was performed after 8 weeks and mandibles were collected to isolate osteoblastic cells as described below.

Cell Culture
The hemimandibles were ground and the alveolar bone fragments were transferred to a sterile centrifuge tube for digestion with collagenase type I (Gibco) at the concentration of 1mg/mL and placed in a water bath at 37 °C under constant stirring. After thirty minutes of digestion, the supernatant was extracted and transferred to another centrifuge tube containing an equal amount of culture medium. This collagenase digestion procedure was performed six times. Supernatants from the first two digests were discarded and the last four were centrifuged at 2000 rpm for 5 min. Cells and fragments were suspended and placed in culture bottles with α-MEM (Gibco), supplemented with 10% fetal bovine serum (Sigma-Aldrich, ST Louis, MO, USA), 10 -7 M dexamethasone (Sigma), 2 mL of gentamicin and 0.3μg/mL fungizone (Gibco). After reaching confluence, cells were enzymatically released from the culture flasks using 1 mM EDTA (Gibco) and 0.25% trypsin (Gibco). Cells were centrifuged at 2000 rpm for 5 min and homogenized in supplemented medium for osteogenic differentiation (MTS) containing α-MEM (Gibco), 10% fetal bovine serum (Sigma), 10 -7 M dexamethasone (Sigma) 5 g/mL ascorbic acid (Sigma), 7 mM β-glycerophosphate (Sigma), 0.3 g/mL fungizone (Gibco) and 50 g/mL of gentamicin (Gibco). The first passage was seeded in 24-well plates at a cell density of 2x10 4 cells per well. Throughout the culture period, the cells were maintained in a humid environment with 5% CO 2 and 95% atmospheric air, and the culture medium was changed every 3 days.

Cell Proliferation
Cell proliferation was assessed by MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) at 7, 10, and 14 days. To this end, cells were incubated with 10% MTT (5 mg/mL) in culture medium at 37 °C for 4 h. The medium was then aspirated from the well, and 1mL of isopropanol (0.04N HCl in isopropanol) was added to each well. The plates were placed on a shaker for 5 min and 200 µL of this solution was transferred to a 96-well plate. The optical density was read at 570 nm (µQuant, BioTek Instruments, Winooski, VT, USA).

In Situ Detection of Alkaline Phosphatase
Analysis of in situ detection of alkaline phosphatase was performed after 7, 10 and 14 days. Cells were washed twice with PBS at 37 °C. There were dissolved 320 mg of Trizma® Pre-set crystals (Sigma-Aldrich) in 20 mL of deionized water. From this solution, 18 mL were added to 2 mL of dimethylformamide (Merck) with 8 mg Naphthol (Sigma-Aldrich). The next step was adding 7 mg of Fast Red reagent (Sigma-Aldrich) to this final work solution. Finally, cells were incubated in 1mL of this solution in a humidified atmosphere at 37 °C with 5% CO 2 for 30 min and then removed from the wells for subsequent qualitative and quantitative analysis.

Mineralized Matrix Formation
Mineralized matrix formation was detected after 17 and 21 days by means of Alizarin Red S (Sigma-Aldrich) staining for areas rich in calcium. Attached cells were fixed in 10% formalin at 4 ºC, for 2 h. After fixation, specimens were dehydrated through a graded series of alcohol, which was followed by staining with 2% Alizarin Red S, pH 4.2, for 10 min. Calcium content was evaluated with a colorimetric method formerly described (11).

Quantitative Gene Expression (Real Time PCR)
To evaluate the quantitative expression of runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alp), osteocalcin (Bglap), osteoprotegerin (Opg), receptor activator of nuclear factor kappa-β ligand (Rankl) and osteopontin (Opn) genes, after 10 days of culture the medium was removed from the wells to add Trizol LS (Life Technologies, Grand Island, NY, USA) at room temperature under agitation. The samples were transferred for eppendorf vials and stored in freezer -20 °C for 48 h. After this period, it was performed RNA extraction utilizing SV Total RNA Isolation System kit (Promega, Madison, WI, USA), following manufacturer's instructions. Following, total RNA was quantified from 1 μL sample inNanoVue equipment (GE Healthcare Life Sciences, Piscataway, NJ, USA) in different wave lenght (260, 280, 230 e 320 nm) and the integrity of each sample was evaluated utilizing a chip able to verify RNA samples in the concentration of 25 to 500 ng/μL, with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). There were considered viable samples those with RIN higher than 8. After RNA integrity confirmation, cDNA was created from 1 μg de total RNA in Mastercycle Gradient (Eppendorf, Hamburg, Germany) by means of reverse transcriptase enzyme, using High-capacity cDNA Reverse Transcription kit (Applied Biosystems, Fort City, CA, USA), following manufacturer's instructions. Table 1

Statistical Analysis
Data were expressed as mean ± standard deviation and analyzed by means of analysis of variance (ANOVA) or nonparametric Kruskal-Wallis, with the level of significance set at 5% (p≤0.05) using the software GraphPad Prism 5.0e. (GraphPad Software, Inc., La Jolla, CA, USA)

Cell Proliferation
Cell proliferation was analyzed at 7, 10 and 14 days with a statistically significant decrease in all periods (p=0.017, p=0.001; p<0.001, respectively) in OVX group compared to sham group (Fig. 1). Figure 2 shows that at 7 days a significant increase in alkaline phosphatase in situ detection was observed in qualitative and quantitative analysis (p<0.001) in OVX Opn

Runt-related transcription factor 2 Rn01512298_m1
Opg Osteoprotegerin Rn00563499_m1 Gapdh Glyceraldehyde 3-phosphate dehydrogenase Rn99999916_s1  group when compared to the sham group. At 10 and 14 days, this activity decreased significantly (p=0.005 and p<0.001, respectively). Figure 3 shows that the evaluation of mineralized calcium nodules showed a significant decrease in the mineralization of cells from osteoporotic group compared to sham group at 17 and 21 days in both qualitative and quantitative analysis (p=0.007 and p=0.007, respectively).

Quantitative Gene Expression
Runx2, Alp and Bglap genes were downregulated in OVX group when compared to sham group (p=0.037, p=0.028 and p=0.028, respectively). The opposite was observed with Opg gene, which showed an upregulation in OVX group when compared to sham group (p=0.028). Rankl and Opn genes did not show statistically significant difference between the groups (p=0.828, p=0.342, respectively) (Fig. 4).

Discussion
In the present investigation there was observed that the model of osteoporosis utilized affected viability, mineralization and gene expression of mandible alveolar bone osteoblasts, evaluated by means of biochemical and molecular assays. We induced osteoporosis by ovary resection, which is a well-known and recognized method to use in animal models (12) and conducted the investigate of possible alterations at 8 weeks after ovariectomy, knowing that each part of the mandible as well as other body parts are expected to show completely different bone changes in response to osteoporosis (13).
The MTT assay showed a lower cell proliferation in OVX group in all experimental periods. These results differ from other investigations that used cells from different sites such as bone marrow, when osteoblastic cells showed higher or similar cell proliferation in OVX groups when compared to sham group (14). Nevertheless, Semeghini et al. (15) also showed lower cell proliferation of calvaria osteoblastic cells from female ovariectomized whereas bone marrow cells from sham and OVX groups had similar proliferation.
Cell differentiation can be evaluated by alkaline phosphatase, which is a bone marker protein that increases and stimulates mineralization (16). In the present investigation, its activity was initially higher for OVX group, which may be explained by a higher energy metabolism caused by a higher generation of ATP that occurs in the presence of osteoporosis (17), suggesting a compensatory function as an important method occurring in chronic diseases like osteoporosis. The same authors also observed a higher Alp activity in mandibular osteoblasts of OVX group. In later periods alkaline phosphatase levels were significative lower for OVX groups, suggesting that in later periods of an osteoporotic condition, Alp activity declines, facilitating osteoclast activation and generating the imbalance of bone formation and resorption (18)(19)(20).
The selected genes for rt-PCR analysis are involved in the modulation of bone remodeling mechanism (21). It was observed that Alp, Bglap and Runx2 genes were downregulated in OVX group, corroborating the reduced extracellular matrix formation observed in the present study. In agreement to Cheng et al. (22), the expression of Runx2 is critical for osteoblast differentiation skeletal morphogenesis and may modulate the expression of early and late essential bone biomarkers in a situation of osteoporosis through overexpression of miRNAs like MiRNA-365a-3. The present results suggest that downregulation of Runx2 significantly downregulated the expression of Alp and Bglap, weakening the capability of mineralization.
We observed a significative higher expression of Opg, in contrast with Rankl and Opn genes that did not show statistically significant difference between the groups. Osteoprotegerin and Rankl are produced by osteoblast lineage cells and represent a key link between bone formation and resorption during bone remodeling. Rankl stimulates osteoclastic activity and differentiation and inhibits osteoclast apoptosis whereas osteoprotegerin inhibits osteoclast formation by binding to Rankl (23). The higher expression of Opg in Ovx groups have also been observed in a previous report (14) with the use bone marrow osteoblastic cell source. In agreement to Yu et al. (17), the removal of the ovaries reduces estrogen concentrations, leading to a need for initial enhancement of osteoblast function in order to compensate for the effects of the decrease in estrogen. Thus, we suggest that the modulation of Opg and Rankl might differ with other time periods or further development of osteoporosis. On the other hand, the lack of difference for Opn expression in the present investigation might be due to the fact that we performed the evaluation with 10 days of culture, possibly not demonstrating its effects in activities such as adhesion and mineralization promoted by osteopontin.
The evaluation of gene expression in one time period could be faced as a limitation of the present study, as well as the possible variability among animals that might exist. A future perspective is to perform in vivo experiments including histological and microtomographic analysis to confirm the present data.
Our results suggest that osteoporosis has a deleterious effect on alveolar bone cells from ovariectomized rats and further in vivo and clinical investigations are thus warranted to confirm this association.