Ovariectomized rats as a model of postmenopausal osteoarthritis: validation and application

Abstract

We aimed to assess the effect of ovariectomy on cartilage turnover and degradation, to evaluate whether ovariectomized (OVX) rats could form an experimental model of postmenopausal osteoarthritis. The effect of ovariectomy on cartilage was studied using two cohorts of female Sprague-Dawley rats, aged 5 and 7 months. In a third cohort, the effect of exogenous estrogen and a selective estrogen receptor modulator was analyzed. Knee joints were assessed by histological analysis of the articular cartilage after 9 weeks. Cartilage turnover was measured in urine by an immunoassay specific for collagen type II degradation products (CTX-II), and bone resorption was quantified in serum using an assay for bone collagen type I fragments (CTX-I). Surface erosion in the cartilage of the knee was more severe in OVX rats than in sham-operated animals, particularly in the 7-month-old cohort (P = 0.008). Ovariectomy also significant increased CTX-I and CTX-II. Both the absolute levels of CTX-II and the relative changes from baseline seen at week 4 correlated strongly with the severity of cartilage surface erosion at termination (r = 0.74, P < 0.01). Both estrogen and the selective estrogen receptor modulator inhibited the ovariectomy-induced acceleration of cartilage and bone turnover and significantly suppressed cartilage degradation and erosion seen in vehicle-treated OVX rats. The study indicates that estrogen deficiency accelerates cartilage turnover and increases cartilage surface erosion. OVX rats provide a useful experimental model for the evaluation of the chondroprotective effects of estrogens and estrogen-like substances and the model may be an in vivo representation of osteoarthritis in postmenopausal women.

Figure 1

Normal levels of CTX-II (collagen type II fragments; μg/mmol, creatinine-corrected) in six male and six female Sprague–Dawley rats. Error bars indicate SEM.

Figure 2

Sections from the knees of 7-month-old rats subjected to ovariectomy, stained with Toluidine blue, showing the distal femur and proximal tibia (a,b) with the meniscus (M) to the left (a). The surface erosion is indicated by the long, thin black bar (b). Scale bars: 200 μm.

Figure 3

Knee sections, stained with Toluidine blue, showing effects of sham operation (a,c) or ovariectomy (b,d) in 7-month-old rats. In (c) and (d), the structure of the collagen fibers is visualized by polarized light. The sham-operated rat (a,c) shows a healthy articular cartilage surface, whereas the ovariectomized rat (b,d) shows surface erosion (b, framed area) and alterations in the structure of the collagen fibers (d, framed area). Scale bars: 200 μm.

Figure 4

Cartilage surface erosion in four condyles in 5-month-old (a) and 7-month-old (b) female rats maintained for 9 weeks after ovariectomy or a sham operation. The erosion (expressed as percentage of total cartilage surface) is presented as mean erosion + SEM for the two groups (OVX and sham-operated). Mean scores are represented for each of the four condyles – medial tibia (Medial T), medial femur (Medial F), lateral tibia (Lateral T), and lateral femur (Lateral F) – and for all four taken as a group (Total). P values indicate difference between ovariectomized (OVX) and sham-operated rats assessed using the nonparametric Mann–Whitney U test.

Figure 5

Severity of cartilage surface erosion in knee-joint cartilage of 5-month-old ovariectomized (OVX) rats treated with the vehicle alone (OVX vehicle), with estrogen (OVX estrogen), or with the selective estrogen receptor modulator (SERM) (-)-cis-3,4-diarylhydroxychromane, given in either a low dose (0.2 mg/kg per day; OVX SERM low) or a high dose (5 mg/kg per day; OVX SERM high). Means for vehicle-treated sham-operated rats are also included (Sham). The erosion is expressed as percentage of total cartilage surface. The left side of the graph shows the accumulated total mean score for all four joint compartments (medial and lateral femur and tibia) and the right side, for the medial femur only. Error bars indicate SEM. The significance of differences between treatment groups and the OVX vehicle group was assessed using Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Cartilage and bone turnover in the ovariectomized (OVX) and sham-treated (SHAM) rats. Cartilage turnover was assessed using collagen type II fragments (CTX-II) as a marker (a,b), and bone resorption was determined by measurement of collagen type I fragments (CTX-I) (c,d). Measurements, made at the weekly intervals shown, are from rats that were (a,c) 5 months old and (b,d) 7 months old at the beginning of the study. Data are presented as average percentage of individual baseline, with error bars representing SEM.

Figure 7

Bone and cartilage turnover in 5-month-old ovariectomized (OVX) rats treated with vehicle alone (OVX vehicle), estrogen (OVX estrogen), or the selective estrogen receptor modulator (SERM) (-)-cis-3,4-diarylhydroxychromane, given in either a low dose (0.2 mg/kg per day; OVX SERM low) or a high dose (5 mg/kg per day; OVX SERM high). Values for vehicle-treated sham-operated rats (Sham) are also included. Bone resorption was determined by measurement of collagen type I fragments (CTX-I) (a), and cartilage turnover was assessed using collagen type II fragments (CTX-II) as a marker (b). Measurements were made at the weekly intervals shown, starting when the rats were 5 months old. The significance of differences between groups was assessed by nonparametric analysis of variance (ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8

Association between cartilage surface erosion (score for total knee) and collagen type II degradation products (CTX-II). Rats from all treatment groups in cohort C (see text and Table 2) were stratified according to CTX-II change after 4 weeks and the average surface erosion for each quartile (Q) is presented. Error bars indicate SEM. P = 0.001 by nonparametric analysis of variance (ANOVA).