CrossRef (0)
AbstractOsteoporosis is a significant health issue in postmenopausal women, causing more than 8.9 million fractures annually. This means an osteoporotic fracture is diagnosed every 3 seconds worldwide (1). Approximately 200 million women suffer from osteoporosis, and its prevalence dramatically increases with age in postmenopausal women (1/10 in their 60s, 1/5 in their 70s, 2/5 in their 80s, and 2/3 in their 90s) (2-4). In Korea, two-fifths of women aged 50 and older have postmenopausal osteoporosis (5).
Mouse models have been extensively used for osteoporosis research through methods such as ovariectomy, loss of estrogen receptor function, dietary interventions, and immobilization (6,7). Among these, ovariectomy has been widely adopted to mimic postmenopausal osteoporosis over the past few decades (5,8-13). Only a few studies have examined the time-course of bone biochemical parameters in ovariectomized mice (14,15), and no studies have established a postmenopausal osteoporosis model induced by bilateral ovariectomy (biOVX) using X-ray imaging, biochemical, and histological analyses. Therefore, the bone loss in a time-course manner has not been fully investigated, even though research on postmenopausal osteoporosis has increased.
Thus, the aims of this study were to: 1) evaluate the effects of biOVX-induced bone loss, and 2) establish a suitable model for studying postmenopausal osteoporosis in animals through dual-energy X-ray absorptiometry (DXA), biochemical analysis of blood serum, and histological evaluation of femoral bone.
Fifty female BDF-1 mice (6 weeks old, Orient Co.) were housed in ventilated cages in a temperature, humidity, and light-controlled environment (11L:13D) and provided food and water ad libitum. The animal care protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University Bundang Hospital (IACUC No.: BA-1910-282-082-01). After a two-week adaptation period, mice were anesthetized with an intraperitoneal injection of 30 mg/kg zoletil (Virbac) and 10 mg/kg Rompun (Bayer). Bilateral ovaries were removed through incisions in the dorsal flank, and the incisions were sutured with 4-0 silk sutures within 5 minutes (16).
Fig. 1 illustrates the experimental design. Briefly, animals were randomly divided into two groups: with or without biOVX. Ovariectomized mice were subdivided into four groups based on the sacrifice day (4, 8, and 12 weeks post-biOVX). Animals were sacrificed by cervical dislocation under IACUC guidelines.
Fig. 1. Comparison of calcium and magnesium in serum measurements. Serum calcium and magnesium were measured to evaluate the effects of bilateral ovariectomy (biOVX) in a time-dependent manner. (A) shows that serum calcium significantly decreased at 4 and 8 weeks after biOVX, but recovered after 12 weeks. (B) illustrates a significant reduction in serum magnesium 4 weeks after biOVX, which recovered 8 weeks later. Asterisks indicate significant differences.
Bone mineral density (BMD), fat distribution, bone mineral content (BMC), and bone volume (BV) were measured using DXA (Inalyzer, Medikors) as previously described (17).
An automatic biochemistry analyzer (Stat Profile Critical Care Xpress, Nova Biomedical) was used to measure the contents of Ca2+ and Mg2+ in whole blood of sham and biOVX groups (sacrificed on 4, 8 and 12 weeks after OVX) as previously described (10).
Femoral bone tissues were obtained from each mouse at the day of sacrifice and then immediately fixed with 4% paraformaldehyde. After 48 hours, tissues were washed with 1X phosphate-buffered saline, and then decalcified by 10% EDTA solution to smoothen the tissue for further progress. Decalcified femurs were then processed and embedded in paraffin blocks. Paraffin sections (5 mm) of each tissue were mounted on slides and stained with hematoxylin–eosin (Merck) as we previously described (18,19).
Data were presented as the mean ± standard error of mean. Statistical difference between groups were presented on raw data by one-way analysis of variance (ANOVA) followed by Turkey’s post-hoc analysis.
As shown in Table 1, biOVX showed a reduction trend in total body weight, body fat and fat in tissue while these criteria had gradually increased compared with 4 weeks after biOXV. However, there were no significant differences between sham and OVX 4-12 groups (Table 1).
A comparison of body weight and fat in total body and tissue from sham control and bilateral ovariectomy groups
| Sham | 4 weeks | 8 weeks | 12 weeks | |
|---|---|---|---|---|
| Body weight (g) | 21.17 ± 0.78 | 18.27 ± 0.59 |
23.38 ± 1.61 |
27.76 ± 2.04 |
| Total body fat (g) | 13.50 ± 3.10 | 8.86 ± 1.07 |
15.86 ± 3.19 | 19.46 ± 5.16 |
| Fat in tissue (%) | 14.00 ± 3.23 | 9.03 ± 1.11 |
16.34 ± 3.28 | 20.01 ± 5.28 |
Values are presented as mean ± standard error of mean.
Asterisks indicate statistically significant differences, respectively. *
With respect to the level of Ca2+ and Mg2 in both sham and ovariectomized mice, biOVX significantly decreased the Ca2+ and Mg2 levels on 4 weeks compared with sham controls. On 8 weeks of biOVX, serum Ca2+ was still significantly lower than sham control while the Mg2 of blood sera from biOVX was comparable with the mice without biOVX. After 12 weeks, serum Ca2+ and Mg2 did not show any significant differences when comparing with sham control as shown in Fig. 2A, 2B.
Fig. 2. Bone health measured by dual-energy X-ray absorptiometry in mice. (A) Bone mineral content, (B) bone mineral density (BMD), and (C) femur volume were significantly reduced 4 weeks after bilateral ovariectomy (biOVX). However, by 16 weeks, all measurements had recovered following biOVX.
Four weeks after biOVX, the level of BMC, BMD, and BV in femur were significantly reduced as Fig. 3 represented. Eight weeks after biOVX, BMD and BV in femur were still significantly lower than those of sham control. However, only femur BMD showed a statistical difference when comparing with sham control.
Fig. 3. Histological evaluation using hematoxylin and eosin staining. Bone health was assessed through histological evaluation. Following bilateral ovariectomy, osteoporotic tissue appeared in a time-dependent manner. Arrows indicate the bone microstructure observed in the histological sections.
Our study demonstrated that bone loss occurred in biOVX mice as early as 4 weeks post-surgery and was sustained for up to 12 weeks. This bone loss was confirmed through X-ray imaging, biochemical analysis of blood serum, and histological examination of femoral microstructure. In 2010, Park et al. (5) had already shown that biOVX causes significant changes in BMD in rats. They also postulated that the OVX rat model they used is suitable for studying issues related to post-menopausal bone loss (20-22). Several studies have evaluated whether ovariectomy-induced osteoporosis occurs by 8 weeks post-biOVX, and Suzuki et al. (23) demonstrated that it is sustained for up to 6 months in a mouse model. Furthermore, the OVX-induced loss of cancellous bone was most pronounced at 8 weeks, moderate at 12 weeks, and only a slight bone loss was observed at 16 weeks compared to the SHAM16 control group across four different strains (C57BL/6, BALB/C, ICR, and Kunming) (11,23,24). Similarly to our findings, Song et al. (24) demonstrated that ovariectomy dramatically induced bone loss, as observed through micro-computed tomography (μCT) parameters in the tibia and femur, as well as through serum biomarkers such as alkaline phosphatase (ALP), osteocalcin (OC), N-terminal propeptide of type I procollagen (P1NP), and C-terminal telopeptide of type I collagen (CTX1), 8 weeks after the removal of bilateral ovaries. Collectively, the combination of bone bioimaging and biochemical analysis of blood sera between 8 and 16 weeks post-biOVX is considered optimal for studying osteoporosis.
Menopause can lead to multiple organ dysfunctions, including osteopenia, osteoporosis, anxiety and depression, obesity, alterations in tissue-specific stem cells, hepatic steatosis, and other conditions (25-29). Women with surgical premature ovarian insufficiency (POI) are at a greater risk for bone loss and fractures compared to naturally menopausal women, likely due to the acute and abrupt onset of estrogen and androgen deficiency (30). Since Albright’s foundational work, the role of estrogen in bone health has been well described over the past decades (31-34). Estrogen plays a role by 1) stimulating bone formation through its direct effect on osteoblasts, 2) regulating osteoclast activity via cytokines, growth factors, and lysosomal enzyme production, and 3) modulating bone metabolism by influencing the expression of various hormones (35). Thus, pharmacological hormone replacement therapy (pHRT) has been widely used for the clinical treatment of osteoporosis in both post-menopausal women and younger women with POI (34,36). Nevertheless, pHRT is associated with various complications, including cardiovascular disease, cognitive dysfunction and dementia, estrogen-related breast and endometrial cancers, thromboembolism, and others, as previously described by Humphries and Gill (37-39). Interestingly, Sittadjody and colleagues proposed that a novel cell-based hormone replacement therapy (cHRT) utilizing three-dimensional bioengineered constructs could be developed, with its efficacy enhanced by the incorporation of bone marrow-derived mesenchymal stem cells (40,41). They also postulated that novel therapeutic approaches for osteoporosis, including cHRT, will be necessary to prevent complications associated with pHRT. Based on these findings, this study not only provides an efficient animal model for osteoporosis research but also expands the potential therapeutic approaches for pHRT-related complications.
Unfortunately, we did not measure sex hormones produced by the ovaries, such as estrogen and progesterone, in the present study. Our analysis focused on bioimaging with DXA rather than μCT, as well as biochemical and histological assessments. Future studies will include measuring osteoporosis-associated biomarkers (e.g., ALP, OC, P1NP, CTX1) and immunohistochemistry. Furthermore, a novel therapeutic approach as an alternative to pHRT for osteoporosis was not explored in this study.
Collectively, we found that biOVX causes time-course bone loss that mimics post-menopausal osteoporosis in older women. Therefore, we strongly suggest that this study provides an efficient mouse model for investigating bone loss and developing pharmaceutical, cell-based, and tissue-based therapies for aged women. However, further studies will be required.
None.
No potential conflict of interest relevant to this article was reported.
This project was financially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2018R1D-1A1B07046419).
Conceptualization: all authors. Formal analysis: DY. Funding acquisition: JL. Investigation: DY. Methodology: DY. Project administration: JL. Supervision: JL. Validation: all authors. Visualization: DY. Writing – original draft: all authors. Writing – review and editing: JL.
