Effect of enhanced masticatory force on OPG, RANKL and MGF in alveolar bone of ovariectomized rats

Abstract Menopause induces oral bone loss, leading to various oral diseases. Mastication importantly affects bone metabolism in the jawbone. Objective: To analyze the effect of enhanced masticatory force on osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), and mechano–growth factor (MGF) in alveolar bone of ovariectomized rats and to study the mechanics mechanism of the alveolar bone of ovariectomized rats response to enhanced masticatory force. Methodology: Thirty Sprague Dawley rats were randomly divided into three groups: sham–operation group (fat around the removed ovary + normal hard diet), model group (ovariectomy + normal hard diet), and experimental group (ovariectomy + high hard diet). It was a 2–month experiment. Enzyme–linked immunosorbent assay (ELISA) detected serum estradiol (E2), osteocalcin (BGP) and alkaline phosphatase (ALP) in rats. Bone histomorphometric indices in the third molar region of maxilla were detected by micro-CT; protein expressions of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Western blot; and gene expression of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Quantitative Real–Time PCR. Results: Comparing with model group, serum E2 in experimental group increased but not significantly, serum BGP and serum ALP in experimental group decreased but not significantly, OPG in experimental group in alveolar bone increased significantly, RANKL in experimental group in alveolar bone decreased significantly, RANKL/OPG ratio in experimental group decreased significantly, MGF in experimental group in alveolar bone increased significantly, bone volume to total volume fraction increased significantly in experimental group, trabecular thickness increased significantly in experimental group, and trabecular separation decreased significantly in experimental group. Conclusion: Enhanced masticatory force affected the expression of OPG, RANKL, and MGF in alveolar bone of ovariectomized rats, improved the quality of jaw bone of ovariectomized rats, and delayed oral bone loss by ovariectomy.


Introduction
Menopause results in oral bone loss, [1][2][3] which can lead to loose tooth, tooth loss, and periodontal disease. [2][3][4] Kribbs 5 (1990) found women with osteoporosis were three times more susceptible for teeth loss than their healthy controls. As reported, approximately 200 million women are affected by osteoporosis worldwide, which includes one third of 60 to 70-year-old women and two thirds of over 80 year-old women. 6 Skeletal osteoporosis results in lower bone mineral density of women's mandibles. 7 Oral bone loss induced by menopause is now a highly discussed topic among scholars.
Currently, three main methods are used to prevent and treat oral bone loss: drugs, surgery and physiotherapy. [8][9][10][11][12][13] Many scholars investigated drugs for oral bone loss. [8][9] A physical method of prevention and treatment, biomechanical therapy has drawn attention of scholars because of its noninvasive and non-side effects. Mavropoulos, et al. 11-13 (2004, 2010, 2014) studied the effects of normal masticatory force on alveolar bone morphology of growing, normal adults and ovariectomized rats. Grünheid,et al. 14 (2011) reported the effects of chewing force on jaw muscle and jaw mineralization in New Zealand white rabbits. Denes, et al. 15

Reagents and instruments
The following reagents and instruments were used:

Grouping and processing
Thirty SD rats were randomly divided into three groups: sham-operation group, model group, and experimental group. Before the experiments, all rats were acclimatized for one week. For the shamoperation group, only little fat around the ovary was removed without ovariectomy, while the other rats were bilaterally ovariectomized using a dorsal approach. One week later, the ovariectomized rats were divided into model group and experimental group. Rats in sham-operation group and model group continued to be fed with normal hard diet, while rats in experimental group were fed with high hard diet.
All rats had free access to food and water. It was a 2-month investigation.

Tissue preparation
All rats were killed two months after fed by grouping. Before this, all rats stopped to eat and kept drinking water for 12-24 hours.

Effect of enhanced masticatory force on serum biochemical parameters in ovariectomized rats
Estrogen is the key hormone to maintain bone mass, 20 and its reduction is the main cause of osteoporosis in postmenopausal women. 21 Estrogen reduction results in microstructural changes in alveolar bone, and alveolar bone resorption is a local manifestation of osteoporosis in the oral field. 22 Animal models for osteoporosis are usually established by removing ovaries to lower estrogen levels. 2,[20][21][22] Osteoporosis is related to oral bone loss in animal studies on rats and it results in lower bone mineral density of women's mandibles. [1][2]7 In this study, when comparing with sham-operated group, serum E2 level in model group and experimental group decreased significantly, indicating ovariectomy was successful.
Compared with model group, serum E2 level in experimental group increased, but not significantly.
Many studies have shown that moderate exercise  The load on jawbone is mainly chewing force, and mastication promotes jaw growth and development.
The evolutionary process of human beings has indicated that chewing force has a great influence on jaws evolution and development. 28  OPG, inhibits the expression of RANKL, which reduces RANKL/OPG ratio, and then promote osteogenesis. 37

Conclusions
In conclusion, our study demonstrated several