The Relationship Between Osteoporosis And Osteoarthritis In Ovariectomized Rats

Huang Chen ¹,  Ren Xu-rong ²,  Wang Tao ³,  Zhang Liu ⁴ .

Abstract:

Objective  Ovariectomy(OVX) can cause bone loss in rats, but little is known about the relationship of osteoporosis(OP) and osteoarthritis(OA) .This study investigated how OA affects the development of OP in OVX rats.  Merterials and Methods  Fifty 3-month-old female Sprague-Dawley rats were divided randomly into five equal groups. The baseline control group (BL group) was euthanized at the beginning of the experiment. A bilateral ovariectomy was performed in 10 rats (OVX group) and another group of 10 rats was subjected to a sham surgery (Sham group). Osteoarthritis was induced by transection of the anterior cruciate ligament of the right knee in 10 rats (OA group). Bilateral ovariectomy and transection of the anterior cruciate ligament of the right knee was preformed in the last group (OVX+OA group). Bone mineral density (BMD) measurement and bone histomorphometric analysis were applied to the right femora in all rats evaluated 6 months after surgery. Before death, they were all double-labeled with subcutaneous injections of tetracycline and calcein.  Results  The bone mass of the OVX group is significantly lower than that of the Sham group (P < 0.05). The bone mass of the OVX+OA group is significantly higher than that of the OVX group. The bone mass of the OVX+OA group is significantly lower than that of the OA group. There is no significant difference in bone mass between the OA and Sham groups. Conclusion  In conclusion, OA retards the development of OP in the distal femur of OVX rats.

J.Orthopaedics 2010;7(1)e3

Keywords:

Osteoporosis; Osteoarthritis; Bone histomorphometry; Ovariectomy

Introduction:

Primary osteoporosis (OP) often occurs in the elderly, especially postmenopausal women. Many risk factors are associated with primary OP, such as smoking, hormone condition, menstrual history, and genetic risk factors. The reduction in estrogen production is thought to be responsible for bone loss in postmenopausal OP patients ¹. Primary osteoarthritis (OA) is a prevalent chronic degenerative joint disease and it most often affects the joints of limbs such as the knee and hip. Some studies have observed that primary OP and primary OA in peripheral joints do not often occur in the same patient, and an inverse relationship was indicated between these two common diseases ^{2, 3}. However, previous studies of the relationship between OP and OA did not exclude the influences of these risk and genetic factors such that these confounding factors possibly influenced the assessment. Therefore, until now, it remains debatable whether OA retards the development of OP^{4, 5}. The use of animal models is a powerful tool for understanding the similarities and differences between them and the human case and for providing preclinical references for the diagnosis and treatment of diseases. Moreover, they can reduce the influences of certain confounding factors that exist in humans but not in experimental animals^6,7.To our knowledge, few animal models have been used for the study of the relationship between OP and OA. Therefore, the purpose of this study was to examine the effects of OA and estrogen deficiency on the distal femoral in OVX rats, widely used as models to simulate the estrogenic condition of postmenopausal women, and to investigate the relationship between the two common diseases in animal model.

Materials and Methods:

Experimental Design

Fifty 3-month-old female Sprague-Dawley rats (Peking University Animal Center), weighing approximately 210±32 g at the beginning of the experiment were randomized into five groups of 10 animals each. The rats were housed in individual cages and were given food and water ad libitum during the experimental period. The rats in the baseline group (BL group) were killed at the beginning of the study. Under 1% pentobarbital sodium 40 mg/kg anesthesia, bilateral ovariectomy was performed on 10 rats through a dorsal approach under sterile conditions (OVX group). Another 10 rats were subjected to sham surgery in which the ovaries were exposed but not removed (Sham group) ^8,9. Osteoarthritis was induced by transection of the anterior cruciate ligament of the right knee in 10 rats (OA group). Bilateral ovariectomy and transection of the anterior cruciate ligament of the right knee in the fifth group(OVX+OA group) ¹⁰. All the rats were sacrificed 6 months after the operation (9 months old). All the animals received fluorochrome double-labeling with subcutaneous injections of 30 mg/kg of tetracycline (Shanghai Xinya Pharmaceutical Factory) and 6 mg/kg of calcein (Sigma Chemical Co. St. Louis, MO) on the tenth day and the third day, respectively, before euthanasia. When soft tissues were carefully removed, the right femur was sawed into 4 equal parts with a low-speed metallurgical saw (Buehler LTD.USA) to take into account of possible differences in the local proportion of trabecular and cortical bone, distal femur, proximal femur, midshaft femur (The two equal regions of diaphysis were considered). The distal parts that we needed were performed BMD measurement with dual–energy X-ray absorptiometry (Norland XR-36, USA) adapted to measuring small objects. Reproducibility, as a coefficient of variation (CV) from five measurements of the same femora after respositioning, was 0.52%. The stability of the instrument was controlled by scanning a phantom two times a week¹¹.

Specimen Preparation and Staining for histomorphometric measurements

The distal parts were trimmed at frontal view to expose the marrow cavity for better fixation and used for the histomorphometric measurement. The distal part of the right femora were fixed with 70% ethanol and then dehydrated in ascending grades of ethanol, defatted in an acetone ethanol mixture (1:1), and embedded in methyl methacrylate without decalcification. The frontal sections were cut at 4 and 8 μm thickness with a microtome (Leica RM 2155, Germany). The 4 μm sections were stained with Goldner’s Trichrome for bone static and cell parameter measurements, and the unstained 8 μm sections were used for dynamic histomorphometric analysis of fluorochrome labeling.

Histomorphometric Analysis

Histomorphometric measurements were performed by an independent individual who was unaware of the experimental protocol, using a digitizing image analysis system (DIAS; KSS Image, Magna, UT, USA) which consists of a light and an epifluorescent microscope coupled to a computer with a morphometry program“stereology”.The measurement site on the bone section was between 1 and 4 mm distal to the growth plate–epiphyseal line and bilaterally between the endocortical envelope. The histomorphometric terminology for cancellous bone measurements was employed according to the methods described by Cui liao et al. ^[12]

Statistical Analysis 

Data were analyzed using the SAS system (Ver. 6.12; SAS Institute, Cary, NC). All descriptive data were expressed as mean ± SD. Student’s t-test and Wilcoxon test for independent nonparametric samples were applied to compare significant differences between the two groups. A two-sided probability value of P < 0.05 was considered statistically significant. The values of the BL group were provided as a reference for comparison but were not included in the histomorphometric analysis.

Results :

Effects of OVX, OA, OVX+OA on distal femoral BMD ( Table 1)

Table 1. BMD values among the five groups (g/cm²)

Data are the mean ± SD of 10 values per group

a p<0.05，significantly different from the time-corresponding Sham group. 

b p<0.05，significantly different from the time-corresponding OVX group. 

c p<0.05，significantly different from the time-corresponding OA group. 

The BMD of the distal femoral in the OVX group increased in comparison with the BL group but significantly decreased compared with the Sham group. There were no differences in BMD between the OA and Sham groups. The BMD of the OVX+OA group were significantly higher than those of the OVX group. The BMD of the OVX+OA group were significantly lower than those of the OA group.

Effects of OVX, OA, OVX+OA on distal femoral metaphyseal histomorphometry ( Table 2&3)

Table 2. Static histomorphometric indices of cancellous bone in the distal femoral in the four groups

Data are the mean ± SD of 10 values per group

a p<0.05，significantly different from the time-corresponding Sham group. 

b p<0.05，significantly different from the time-corresponding OVX group. 

c p<0.05，significantly different from the time-corresponding OA group. 

Table 3. Dynamic histomorphometric indices of cancellous bone in the distal femoral in the four groups

Data are the mean ± SD of 10 values per group

a p<0.05，significantly different from the time-corresponding Sham group. 

b p<0.05，significantly different from the time-corresponding OVX group. 

c p<0.05，significantly different from the time-corresponding OA group. 

Compared with the Sham group, some static histomorphometric indices of cancellous bone in the OVX group were significantly lower (trabecular bone volume and trabecular number), whereas indices of trabecular separation, osteoclasts number–bone volume referent, and percent osteoclast surface were significantly higher. Similarly, while some dynamic indices were significantly higher than in the Sham group (indices of mineralizing surface–bone surface referent and bone formation rate), the double-labeled perimeter was significantly lower. There were no significant differences in all the histomorphometric indices between the OA and Sham groups. The Tb.Sp, Oc.No/BV, Oc.Pm/BS, MAR, BFR/BS, BFR/BV of the OVX+OA group were significantly lower than those of the OVX group, but the BV/TV, Tb.N, dL.Pm were significantly higher. The Tb.Sp, Oc.No/BV, Oc.Pm/BS, MAR, BFR/BS, BFR/BV of the OVX+OA group were significantly higher than those of the OA group, but the BV/TV, Tb.N, dL.Pm were significantly lower.

Figure 1: The surface of the distal femoral in the Sham group (9 months old)  was very smooth, had the gloss, no articular cartilage destruction and no osteophyte formation on the edge of the femoral condyle. 

Figure 2: The surface of the distal femoral in the OA group (9 months old)  was much coarser than that in the Sham group. There were articular cartilage destruction and osteophytes formation on the edge of the femoral condyle. The degeneration of the knee indicated formation of the OA.

Figure 3: These pictures showed the structures of metaphyses, trabecular bone and epiphyseal line et.al. on distal femoral in Sham group, OVX group, OA group and OVX+OA group rats. The white line on the upper part of the picture was epiphyseal line, the green funicular structures below that were trabecular bone. The measurement site on the bone section was between 1 and 4 mm distal to the growth plate–epiphyseal line and bilaterally between the endocortical envelope. The pictures showed that the trabecular number of the OVX group was significantly lower than that of the Sham group, the trabecular number of the OVX+OA group was significantly higher than that of the OVX group, the trabecular number of the OVX+OA group was significantly lower than that of the OA group (Masson-Goldner Trichrome staining 4 μm sections without decalcification, original magnification 10×).

Discussion :

In our data the rats had remarkable bone loss and high bone turnover 6 months after the ovariectomy. The OVX-induced bone loss was due to an increase in bone resorption caused by estrogen deficiency accompanied by an increase in bone formation that was insufficient to compensate for the increase in resorption thereby leading to bone loss. Our results demonstrated that we duplicated a postmenopausal OP animal model ¹³.

Joint instability was a well known cause of secondary OA of the human knee. To study the pathogenesis of instability induced OA, animal models were frequently used. There was a consistent relationship between the development of OA and knee instability as a result of transection of the anterior cruciate ligament in knees of animal models such as the rat^[10]. In this study, the surfaces of the distal femoral in OA group were much coarser than that in the non-OA group. The degeneration of the knee indicated formation of the OA.

In clinical application, there were three different viewpoints on the relationship between OP and osteoarthritis OA^14,15. The first was that OA retards the development of OP. The second was OA accelerates the development of OP. The third was these two diseases were not correlative in pathology process.

This study demonstrated that: 1)The bone mass of the OVX+OA group is significantly higher than that of the OVX group. There was a decrease in bone formation related parameters such as MAR. The bone resorption parameters as Oc.No/BV, Oc.Pm/BS and bone turnover (BFR/BV) were also decreased. The OVX+OA group rats could prevent the OVX-induced cancellous bone loss by a decrease in bone turnover. Our findings suggested that OVX+OA group rats created a positive bone balance compare with the OVX group rats. 2) The bone mass of the OVX+OA group was significantly lower than that of the OA group. There was an increase in both the bone resorption related parameter such as Oc.No/BV, Oc.Pm/BS and bone formation related parameter as MAR. Due to the bone turnover parameter (BFR/BV) in the  OVX+OA group was significantly higher than that of the OA group, the cancellous bone mass were lossed.3)There was no significant differences in bone mass between the OA and Sham groups. That was because the bone formation and resorption parameters in the OA and Sham groups have no difference.

In other clinical research, Verstraeten et al.⁴ found that the osteoarthrotic patients had fewer forearm and other fractures compared with the OP patients, while the OP patients had a significantly lower degree of osteoarthrosis in the hand, hip joints, and spine compared with the osteoarthrotic patients. Therefore, we hypothesize that primary OA might have a protective effect on the progression of OP with its related factors, such as high levels of insulin like growth factor, being overweight, decreased bone turnover, and genetics.

In summary, OA retards the development of OP on the distal part of the femur in OVX rats.

Conclusion:

Reference :

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13. Kalu DN  The ovariectomized rat model of postmenopausal bone loss. Bone and Mineral 1991; 15:175-192

14. Ding RK, Sun CJ, Wang WC  Relationships of the osteoarthritis of the knee joint with the osteoporosis. Hunan Medical Journal 1997; 14:323-324

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This is a peer reviewed paper 

Please cite as: Huang Chen: The Relationship Between Osteoporosis And Osteoarthritis In Ovariectomized Rats

J.Orthopaedics 2010;7(1)e3

URL: http://www.jortho.org/2010/7/1/e3

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