MODERATE PHYSICAL ACTIVITY IMPROVES RAT BONE ULTRASTRUCTURE IN EXPERIMENTAL OSTEOPOROSIS

Abstract

Context and objective

In this study, we aimed to investigate how moderate physical activity improves the bone ultrastructural parameters in rats with glucocorticoid-induced secondary osteoporosis.

Animals and Methods

Research has been carried out on Wistar female rats. Secondary osteoporosis was induced through daily i.m.1.5 mg/kgbw methylprednisolone, over a period of 30 days. A group of rats with induced secondary osteoporosis were subjected to physical activity (swimming) for one hour/day for 30 days. Rats were sacrificed 24 hours after the last administration and femoral bones were used for electron microscopy analysis.

Results

The ultrastructural findings obtained from the rats with osteoporosis showed varying degrees of alteration in all cellular components. A moderate physical effort led to the overall maintenance of the normal ultrastructure of the cells and connective components, protecting the lamellar structure of the compact bone from the deleterious effects of glucocorticoid. The shape and components of osteocytes were also preserved and the accumulation of lipids in the bone marrow diminished.

Conclusions

Physical exercise has been shown to have a protective role by lowering the development of structural alterations specific to osteoporosis. Therefore, moderate physical exercises are recommended for improving the structure of the bone mass affected by glucocorticoid treatment.

INTRODUCTION

Osteoporosis is a metabolic disorder characterized by the progressive loss of bone mineral content and mineral density, which causes the bones to become more porous and to be exposed to fractures (1). Over the past 4-5 decades, demographic growth has led to an undeniable phenomenon of population aging, especially in developed countries (2). Global statistics confirm the aging of the world’s population (3). Thus, the average monthly gain of the elderly population has increased from 795,000 people in 1991 to 847,000 people in 2010.

Osteoarticular diseases, including osteoporosis, have high incidence rates, severe evolutions and unfavourable prognoses; they represent “a major burden” for the public health and social welfare systems, the costs being exorbitant (4). Unfortunately, in 10-15 years’ time, the incidence of osteoarticular diseases will grow in parallel with the extension of longevity (by about 200% among men and by 180% among women past the age of 50)(5).

Over the past 100 years, modern life style has become more sedentary, with a tendency to lower exposure to physical effort and subsequently increased frequency of functional cardiovascular, blood and osteoarticular disorders (6).

Osteoporosis can be classified as primary or secondary. Primary osteoporosis may occur in both genders and at any age, but it tends to manifest in post-menopausal women and in elderly men. By contrast, secondary osteoporosis occurs as a side effect of certain medications, mainly with corticosteroids, or of endocrine diseases (e.g. hypogonadism). Long-term glucocorticoid medication, as in the case in rheumatoid arthritis and obstructive pulmonary diseases, can induce osteoporosis and increase the risk of fractures (7). For example, in a group of patients who were treated with 10 mg of prednisone for 20 weeks, bone mass density decreased by 8%. Experts have estimated that any patient who is administered a 5 mg or higher oral dose of prednisone for more than 2 months is exposed to a high risk of bone mass loss and, hence, to the occurrence of fractures (8, 9).

Nowadays, special attention is paid to preventing osteoporosis. Among these means, physical activity is considered to have a beneficial effect, if practiced throughout life, on maintaining good bone mass (10). It has been shown that regular physical exercise leads to an improvement by 1% of the bone mineral density in comparison with situations in which physical activities are not carried out (11). This percentage does not appear to be significant, but in time, substantial effects on the quality of bone become apparent. On the other hand, physical activities have a positive effect on neuromuscular functions, enabling the individuals to have better co-ordination, improved balance and greater strength. All these decrease the risk of a fall and bone fracture. Studies conducted on professional athletes have found a real improvement in the bone tissue quality, even after they ceased practicing their sports (12-14). Implementation of regenerative therapy by using mesenchymal stem cells to treat or relieve the state of osteoporosis has had promising results (15). It has been reported a mutual, inverse relationship between osteoblastogenesis and adipogenesis, which can explain the process of bone formation decrease with the onset of osteopenia and the concomitant increase of adipose tissue in the bone marrow (16). It is well known that glucocorticoids can steer the pluripotent cells in the bone marrow towards being transformed into adipocytes instead of bone-forming osteoblasts (17).

In light of the data presented above, we aimed to investigate how moderate physical activity improves the bone ultrastructural parameters in rats with glucocorticoid-induced secondary osteoporosis.

ANIMALS AND METHODS

Experimental design

Female Wistar rats aged 2 years were used, corresponding to the human age of 52 years and weighing 250±10 g at the beginning of the experiment (18). Females are more prone than males to develop secondary osteoporosis around this age, because rat bone gradually moves from modeling to remodeling, just as human bone, this transition being age-related and also induced by major hormonal modifications. Experimental procedures were approved by the Ethical Committee of the university.

Rats were divided into four groups as follows (10 rats/group): control group (C group) fed on balanced standard diet and water ad libitum; a group of rats subjected to physical activity/swimming (S group) one hour/day for 30 days; a group of rats subjected to glucocorticoid-induced secondary osteoporosis (O group) by intramuscular administration of 1.5 mg/kg bw/day methylprednisolone, over a period of 30 days; a group of rats subjected to glucocorticoid-induced secondary osteoporosis (i.m. injection of 1.5 mg/kg bw/day methylprednisolone for 30 days) and physical activity/swimming 1 hour daily for 30 days (OS group). The selection of glucocorticoid dose was based on previous published reports, related to experimental model of osteoporosis (19, 20). Swimming exercises were performed by rats according Ooi et al. (21) method. We chose rats because they are excellent animal models for studying bone health due to the similarity in terms of morphometry and structure with human bone (21).

Rats were sacrificed 24 hours after the last administration and femoral bones were used for transmission electron microscopy analysis. Samples were collected from the central part of diaphysis. Compact bone was the tissue of election because most of the existing literature reports modifications of the trabecular tissue in osteoporosis and fewer data are available concerning compact bone.

Electron microscopy

Electron microscopy samples were prefixed with 2.7% glutaraldehyde in 0.15 M phosphate buffer at 40C for 5 h, washed in 0.15M phosphate buffer (pH 7.2) and postfixed in 2% osmic acid in 0.15M phosphate buffer, pH 7.2, at 4°C for 2 hours (22). Dehydration was performed in acetone and bone samples were embedded in the epoxy resin Epon 812. Thick sections of 50-70 nm were cut with Leica EM UC6 ultramicrotome and analyzed with a TEM JEOL 1010 electron microscope. The selected images were acquired using a Mega View III camera.

RESULTS

The ultrastructure of the compact femoral bone was normal in the control group (Fig. 1), showing a regular aspect with a parallel-concentric layout of lamellae (Fig. 1A) made of collagen fibres arranged in fascicles or clusters of adjoined bundles, which ensure the relative individuality of each lamella (Fig 1B). Osteocytes located in the lacunae exhibited normal structure, well-structured nucleus with heterochromatin and euchromatin, having a well-defined nucleolus, normally structured, and discretely arranged cellular organelles in the cytoplasm (Fig. 1C). In the control group there is a membrane separation between bone marrow and the compact bone border (Fig. 1D), therefore the wall of a lamella borders directly the thin sheet that delineates the marrow. The bone marrow contains numerous hematopoietic cells (Fig. 1E), the most prominent cells being megakaryocytes (Fig. 1F).

Figure 1.

Ultrastructural aspects of control compact bone. A: Parallely disposed lamellae (bar – 10 μm); B: Collagen fibrils (bar – 2 μm); C: The osteocyte (bar – 2 μm); D: The limit between the lamellar system and bone marrow (bar – 10 μm); E: Bone marrow cells (bar – 10 μm); F: A megakaryocyte in bone marrow (bar – 5 μm).

There were no differences between the control group and S group in terms of the number of lamellae in an osteon and of the width between the lamellae, or of the lamellar connective-bone structure of the collagen (Fig. 2A). Osteocytes had a structure similar to the control, but there was a higher incidence of canaliculi between the osteocytes (Fig. 2B), which suggests an intensification of nutrient circulation between the osteocytes, based on a slight metabolism increase at this level. The limit between bone marrow and lamellar bone was still evident (Fig. 2C), but the structure of marrow cell population was slightly modified compared with the control, with an increasing lymphocyte population (Fig. 2D). Normally structured megakaryocytes were present (Fig. 2E).

Figure 2.

Ultrastructural aspects of compact bone in rats subjected to swimming (S group). A: Parallel disposition of the lamellae (bar – 5 μm); B: The osteocyte (bar – 2 μm); C: The limit between the lamellar system and bone marrow (bar – 10 μm); D: Bone marrow with many lymphocytes (bar – 5 μm); E: A megakaryocyte in bone marrow (bar – 5 μm).

The bone ultrastructure of the O group showed evident changes, as compared with the control (Fig. 3). The lamellar bone was disorganized, no longer being arranged in parallel structures and lysis vesiculations were present in all collagen bundles (Fig. 3A, B). At higher magnifications, it could be noticed that, due to the structural alteration of collagen, the bundles that form the walls of the bone lamellae became thinner (Fig. 3C), which greatly decreases the resistance of the compact bone, making it brittle and incapable of resisting mechanical pressures. Because of collagen structure alterations, many of the canaliculi between the osteocytes were destroyed and thus the supply of nutrients to osteocytes was impaired. Consequently, the structure of the osteocytes was also affected. Thus, vacuolizations of the cytoplasm (Fig. 3D) were followed by the tearing of the surrounding plasma membrane and the lysis of the cytoplasmic content, the picnosis and destruction of the nucleus (Fig. 4A). Many osteocytes eventually turned into lacunae without cellular content (Fig. 4B). The lamellae that delineate the bone marrow also showed an altered structure, as of the neighbouring marrow cells (Fig. 4C). Marrow cell population was either rarefied (Fig. 4D), or showed cells with altered structure, lipid overload and detritus of some destroyed cells (Fig. 4E). Changes also occurred in megakaryocytes: nuclei had an altered structure and the cytoplasm was rarefied, suggesting that thrombocytopoiesis was affected (Fig. 4F).

Figure 3.

Ultrastructural aspects of compact bone in rats with corticoid-induced secondary osteoporosis (O). A: Vacuolisations in the lamellae (bar – 2 μm); B: Altered lamellar system (bar – 2 μm); C: Altered structure of collagen (bar – 5 μm); D: Injured osteocyte (bar – 2 μm).

Figure 4.

Bone ultrastructure in rats with experimentally-induced osteoporosis (O group). A: Vacuolisation of osteocytes (bar – 2 μm); B: Destroyed osteocyte (bar – 2 μm); C: Altered lamella lining the bone marrow (bar – 10 μm); D: Modified cells of the bone marrow (bar – 5 μm); E: Accumulation of lipids in the bone marrow (bar – 5 μm); F: Altered megakaryocyte (bar – 5 μm).

No major ultrastructural changes occurred in OS group, as compared to the control, but minor influences of the glucocorticoids were still visible. The lamellar structure of the compact bone was preserved (Fig. 5A); however, the collagen bundles that form the walls of the lamellae, and also the inter-lamellae collagen, contained small lysis vacuolisations which led, in some areas, to the thinning of collagen walls (Fig. 5B).

Figure 5.

Ultrastructural aspects of the compact bone in rats treated with methylprednisone and subjected to swimming (OS). A: Lamellae and osteocytes with normal appearance (bar – 10 μm); B: Lamellae of different widths (bar – 10 μm); C: Osteocyte with normal structure (bar – 2 μm); D: Normal osteocyte with its canaliculi (bar – 2 μm); E: Unmodified limit between lamellar system and bone marrow (bar – 20 μm); F: Bone marrow containing normal osteoprogenitor cells (bar – 5 μm); G: Unaltered megakaryocyte (bar – 10 μm).

Osteocytes are present between the bone lamellae, with a normal structure of the nucleus and the cytoplasm (Fig. 5C, D). There were fewer canalicular extensions between the osteocytes, also illustrating the negative influence of glucocorticoids. We also noticed a slight decrease of the bone marrow cellular population, even though in the area close to the compact bone lymphocytes were in high number (Fig. 5E). Otherwise, the vast majority of marrow cells had a normal structure (Fig. 5F, G), comparable to the one seen in the control group.

DISCUSSION

Clinical evidences showed that approximately 30–50% of patients receiving chronic glucocorticoid therapy exhibit fractures (23). Therefore, finding of the new methods to prevent osteoporosis progress following treatment with glucocorticoids are useful.

In our study, we aimed to investigate how moderate physical activity improves the ultrastructure of femoral bones in rats with glucocorticoid-induced secondary osteoporosis. Overall, our TEM results revealed that moderate physical effort (swimming) led to the maintenance of the regular ultrastructure of the osteocytes and connective components of the bone, protecting the lamellar structure from the deleterious effects of glucocorticoid administration in rats.

The collagen fibrils are crucial in maintaining the structure and function of the bones. In pathological conditions, such as glucocorticoid-induced secondary osteoporosis, abnormalities occur in both the arrangement and diameter of the collagen fibrils, leading to decreased bone strength and increased risk of fracturing (24-27), which is in agreement with our ultrastructural findings.

Osteoporosis is characterized by reduced bone mass and disruption of cancellous bone architecture (28, 29). From the result of TEM, we observed that most of the osteocytes in O group suffered changes and lamellar bone disorganization. In this regard, it has been confirmed that apoptosis was an important pathological phenomenon involved in the glucocorticoid-induced osteoporosis (30). LoCascio et al. (31) clinical results suggest that long-term glucocorticoid therapy causes a reduction of bone turnover and bone loss occurs predominantly within the first 6 months of treatment.

Previous studies found that there is a mutual relationship between osteoblastogenesis and adipogenesis, which can explain the process of decreased bone formation with the onset of osteopenia and the increase of adipose tissue in the bone marrow (16). Glucocorticoids act by decreasing the capacity of bone formation: osteoblasts are transformed into adipocytes and lipids accumulate in the bone marrow (16, 32). Ultrastructural studies have proved that the decrease of bone mass in SAMP6 mice was due mainly to a lower functioning of the osteoblasts (33). The result is the fact that the process of hematopoiesis, and especially that of osteoblastogenesis are profoundly altered (34).

Certain results have been reported in animal studies on the effects of swimming exercise on bone. For example, it was found that 8 weeks of swimming exercise at 90 minutes per day for 8 weeks enhanced bone mineral density, geometry, and microstructure in femur bones of the ovariectomized rats (21). Other data, confirm that physical exercise contributes to keeping bone mass in good condition both in pre- and in postmenopausal women (11, 35-36).

There is increasing evidence that moderate physical exercise might regulate bone remodeling through osteocytes. It was reported that exercises prevent osteocyte apoptosis and improve some of the microarchitectural parameters (37), increase bone mineral density and osteocyte lacunar occupancy (38) in rats, which is in agreement with our findings.

Our ultrastructural results are in agreement with other studies on the relationship between swimming and bone mass in rats (39) and complementary with clinical research conducted in population-based cohort of women and men, where physical activity was found to be an important modifiable risk factor for bone mineral density (40).

In conclusion, our results suggest that physical exercise (i.e. swimming) may protect against glucocorticoid-induced osteoporosis, although further studies in a more quantitative biochemical and molecular data are required.

Conflict of interest

The authors declare that they have no conflict of interest concerning this article.