Introduction

Dyslipidemias are among the most commonly detected and treated chronic conditions. They are classically characterized by abnormal serum levels of cholesterol, triglycerides, or both, involving abnormal levels of various related lipoproteins. The clinical consequence most commonly associated with dyslipidemia is the increased risk of atherosclerotic cardiovascular disease, which is associated with increased cholesterol and triglyceride levels1.

The two most clinically relevant plasma lipids are cholesterol and triglycerides. Among the physiological roles of cholesterol, it was found to be its presence as a constituent of the cell membrane, precursor of the synthesis of steroid hormones, bile acids and oxysterols, respectively modifier of neuronal signaling molecules. Triglycerides, on the other hand, are a source of energy for muscles and fat tissues2.

From a global perspective, the use of lipid-lowering drugs in high-risk individuals has been recommended by all relevant cardiovascular disease prevention guidelines issued by professional societies in different countries or regions. The increasing availability and usefulness of statins have greatly contributed to reducing the burden of atherosclerotic cardiovascular disease related to hypercholesterolemia3,4,5.

Both hyperlipidemia and osteoporosis are widespread conditions and represent major public health problems worldwide. 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, collectively known as statins, are commonly used for the treatment of hypercholesterolemic patients to reduce cardiovascular risk6,7,8,9.

In recent years, there has been a growing interest in other potential effects and possible benefits of statins other than simply lowering the serum cholesterol. These include the so-called pleiotropic effects, and the possible effect of statins on bone tissue is of particular attention10,11. Studies in the literature have indicated that statins can stimulate osteogenesis and provide therapeutic benefits to patients with osteoporosis12,13,14,15. Their potential as robust modulators of bone healing and inflammation was later revealed, demonstrating their ability to stimulate osteoinduction and angiogenesis – key phenomena for bone reconstruction16,17. It also exhibits the ability to limit bone tissue loss by inhibiting both osteoclastogenesis and osteoblast apoptosis18,19,20. Lipophilic statins, especially simvastatin, lovastatin, and atorvastatin, appear to be the most effective in preventing osteoporosis and supporting the healing of fractures and bone defects21,22.

Fibrates are proliferator-activated receptor (PPAR)-α agonists, used especially in patients with hypertriglyceridemia. They are moderately effective agents in reducing plasma triglycerides and, to a lesser extent, cholesterol23. Regarding bone metabolism, preclinical data have demonstrated that fibrates and, in particular, fenofibrate promote the expression of the BMP-2 gene, thereby stimulating osteoblast differentiation24,25. Fenofibrate has been shown to maintain bone mineral density and whole-body bone architecture in ovariectomized rats, compared to pioglitazone26.

Considering the dual therapeutic potential of the hypolipidemic drugs simvastatin and fenofibrate, as evidenced by literature data, the study aimed to provide valuable and complementary information to evaluate how they influence the evolution of osteoporosis and bone fragility.

The hypothesis of the study was based on the use of minimal doses of simvastatin and fenofibrate to prevent the occurrence of adverse effects and to increase adherence to treatment for long-term administration. This approach is based on the fact that hypolipidemic medication for the prevention of cardiovascular disease is administered on a long-term basis and the effect on bone tissue should be evaluated in this context.

Results

Figure 1a shows the self-diffusion coefficient, D distribution function of water in the femur of rats treated with simvastatin, illustrating the presence of small, medium and large pores in various poses. At week 14 of treatment, there is a tendency to migrate from small pores to medium-sized pores. The peak associated with large pores has a small representation from a quantitative point of view. The tendency of small pores to migrate towards medium-sized pores cannot be directly correlated with the duration of simvastatin administration, but rather with the biological evolution of bone tissue towards osteoporosis. The central values (maximum probability) of the self-diffusion coefficient corresponding to bone measurements for rats treated with statins at 14 weeks of age can also be observed (see also Table 1).

Figure 1b highlights the 2D T2-T2 molecular exchange maps measured for euthanized rats at 14 weeks after initiation of simvastatin treatment. It is highlighted that the area of the peaks located on the main diagonal is significantly increased, which means that there are an increase of fluid molecules retaining their position in the same pores, thus showing a reduced connectivity between the trabecular spaces. The presence of extra-diagonal peaks suggests a connectivity of the pores with a communication from small pores to larger pores.

Within this map, another important aspect it can be observed. This is the fact that there are arrows that indicate a position on the main diagonal where there is no peak, suggesting an extreme value of T2, a phenomenon explained by the fact that at small amounts of water is located on open pores on the bone’s surface and the exchange is mostly present as evaporation.

Within the group of rats treated with fenofibrate, variability in terms of peaks corresponding to pores of different sizes can be observed. The NMR measurement performed on rat femur euthanized on week 14 shows a trend of overlapping peaks associated with two types of medium pores, respectively a small peak representing of the water molecules in large pores (see Fig. 1c). The disappearance of the small pores but the presence of two groups of medium pores emphasizes a natural osteoporotic evolution correlated with the age of the experimental animals. The central values (maximum probability) of the self-diffusion coefficient corresponding to bone measurements for rats treated with fibrates at 14 weeks of age can also be observed (see also Table 1).

Figure 1d shows the 2D T2-T2 molecular exchange maps measured for rats sacrificed at 14 weeks after initiation of fenofibrate treatment. Multiple (2D) peaks located on the main diagonal are highlighted, presenting a significant area, which. However, there are also a series of extra-diagonal peaks, which suggests that the presence of a certain exchange of molecules exists via an implicit connectivity of the pores showing also a preponderant communication from smaller bone pores to larger pores.

Figure 2a show that in the week 16 the separation of small pores from medium pores from rat femur treated with statins is highlighted. There one can observe, a minor representation of small pores, respectively the individualization of medium-sized pores, forming a single peak. Large pores can still be identified, while in the 18th week they will shrink (see Fig. 3a). The central values (maximum probability) of the self-diffusion coefficient corresponding to bone measurements for rats treated with statins at 16 weeks of age can also be observed (see also Table 1).

Fig. 1
figure 1

The distribution of self-diffusion coefficient as evaluation of bone pore sizes in groups of rats treated with (a) simvastatin and (c) fenofibrate, respectively. The 2D T2-T2 molecular exchange maps measured for rats treated with (b) simvastatin and (d) fenofibrate, sacrificed at 14 weeks after the start of treatment. The colors in the 2D T2-T2 molecular exchange maps represents the probability that certain T2,idirect- T2,direct pair of transverse relaxation times characterized the bone pores. The red color meaning a lower probability, while a blue color meaning a high probability.

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The same trend of osteoporotic evolution can be observed, but with the preservation of cytoarchitectonics in terms of the presence of the 3 types of pores, small, medium and large, this effect may be associated with simvastatin treatment.

Fig. 2
figure 2

The distribution of self-diffusion coefficient as evaluation of bone pore sizes in groups of rats treated with (a) simvastatin and (c) fenofibrate, respectively. The 2D T2-T2 molecular exchange maps measured for rats treated with (b) simvastatin and (d) fenofibrate, sacrificed at 16 weeks after the start of treatment. The colors in the 2D T2-T2 molecular exchange maps represents the probability that certain T2,idirect- T2,direct pair of transverse relaxation times characterized the bone pores. The red color meaning a lower probability, while a blue color meaning a high probability.

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In rats treated with simvastatin for 16 weeks, a slight reduction in connectivity between trabecular spaces may be observed as can be seen from the T2-T2 exchange maps presented in Fig. 2b. Peaks on the main diagonal are highlighted, with significantly increased area compared to extra-diagonal peaks, which means that most of the fluid molecules remain in the same pores. There are also some extra-diagonal peaks with significantly reduced area compared to the group treated with fenofibrate, which show a preponderance of connectivity with a communication from small pores to large pores.

Fig. 3
figure 3

The distribution of self-diffusion coefficient as evaluation of bone pore sizes in groups of rats treated with (a) simvastatin and (c) fenofibrate, respectively. The 2D T2-T2 molecular exchange maps measured for rats treated with (b) simvastatin and (d) fenofibrate, sacrificed at 18 weeks after the start of treatment. The colors in the 2D T2-T2 molecular exchange maps represents the probability that certain T2,idirect- T2,direct pair of transverse relaxation times characterized the bone pores. The red color meaning a lower probability, while a blue color meaning a high probability.

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The overlap of medium pores (observed in week 14 for the rats treated with fibrate) seems to be resolved as one can see the distribution of self-diffusion coefficient, D measured for the rats’ femur sacrificed in week 16, which is migrating into a well-individualized medium pore category, along with the constant presence of large pores (Fig. 2c). An impairment of the cytoarchitectonics of bone tissue can be observed, due to the disappearance of small pores. The medium sized pores, which were grouped in two sizes at 14 weeks of age, have shrunk into a single category of medium sized pores. The central values (maximum probability) of the self-diffusion coefficient corresponding to bone measurements for rats treated with fibrates at 16 weeks of age can also be observed (see also Table 1).

Figure 2d shows 2D T2-T2 molecular exchange maps measured for sacrificed rats at 16 weeks after initiation of fenofibrate treatment. In this case, many peaks are located extra-diagonally, which represents a significant connectivity at the level of the trabecular spaces. This change of fluid molecules is achieved in a quasi-balanced way, given that they produce communication from small pores to large pores, and to the same extent they also communicate from large pores to small pores.

At week 18 after the start of simvastatin treatment (Fig. 3a) the peak from the D-distribution associated with small pores becomes visible, being the most quantitatively expressed compared to the other weeks. Medium-sized pores remain predominant, with a slight increase compared to previous weeks. A regression of the degree of cytoarchitectonic bone tissue damage can be observed by the reappearance of a group of small pores and a predominance of medium-sized pores. The central values (maximum probability) of the self-diffusion coefficient corresponding to bone measurements for rats treated with statins at 18 weeks of age can also be observed (see also Table 1).

Figure 3c shows a trend of separation of medium-sized pores, with the appearance of a peak corresponding to small pores in the rats’ femur treated with fibrates and the constant presence of the peak associated with large pores. The beneficial effect of treatment with fenofibrate can be observed due to the reappearance of small pore size, this demonstrating the regaining of a cytoarchitectonic necessary to maintain the function of bone test resistance. This is extremely important because it occurs with long-term treatment, which also occurs in clinical cases. The central values (maximum probability) of the self-diffusion coefficient corresponding to bone measurements for rats treated with fibrates at 18 weeks of age can also be observed (see also Table 1).

Figure 3b highlights the 2D T2-T2 molecular exchange maps measured for sacrificed rats at 18 weeks after initiation of simvastatin treatment. The batches of rats treated with simvastatin (Fig. 3b) and fenofibrate respectively (Fig. 3d) show approximately identical peaks’ organization in the T2-T2 molecular exchange maps, with a small peak at the level of the main diagonal, and multiple small peaks with reduced area present extra-diagonally, suggesting an increased connectivity between pores which present a communication from both large pores to small pores, and from small trabecular spaces to large trabecular spaces.

Discussion

The study confirmed the initial hypothesis, demonstrating by the Nuclear Magnetic Resonance (1H NMR) technique that the lipid-lowering treatment affects the self-diffusion coefficient of water molecules, the results being correlated with the duration of treatment administration27. The evaluation of the effect of lipid-lowering medication on bone tissue should be approached with increased attention to detail. These details include the duration and type of lipid-lowering medication administered26. The study highlighted that the administration of lipid-lowering drugs influences the organic structure of bone tissue, having a potential impact on bone structure, observed by changes in pores at the level of the proximal extremity of the femur of rats.

Table 1 The center values (maximum probability) of self-diffusion coefficient, D, area under the peak and the width at half height expressed in logarithmic scale, by numeric deconvolution of the distribution of self-diffusion coefficient corresponding to the measurement of bones for rats treated with statins and fibrates at an age of 14, 16 and 18 weeks.
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Administration of fenofibrate caused the disappearance of small pores in all 3 stages of evaluation, highlighting a tendency to form two groups of medium-sized pores at 18 weeks. Both simvastatin and fenofibrate affect the cytoarchitecture of bone tissue28,29,30. However, the negative osteoporotic alteration of bone tissue cytoarchitecture is less severe in the case of simvastatin than in the case of fenofibrate.

On the other hand, the study also demonstrated that the use of 2D 1H NMR T2-T2 exchange maps allows a detailed evaluation of osteoporosis from the perspective of pore connectivity. The study demonstrated how lipid-lowering medication, represented by simvastatin and fenofibrate, can influence the biology of bone tissue.

The osteoprotective potential of a medication would consist in the preservation of a bidirectional communication both from larger to smaller pores and in the opposite direction, and this communication should be limited, this being evidenced by a predominance of pores that do not present molecular exchange.

In the groups of the studied rats, a negative influence of lipid-lowering medication in terms of bone cytoarchitecture could be observed by its interference with the biology of bone tissue depending on the duration of administration. Thus, in the short term, both fenofibrate and simvastatin reduce pore connectivity at 14 weeks of treatment. Correlating the data obtained from the recording of two-dimensional T2-T2 exchange maps with the evidence from 1H NMR diffusiometry, it can be concluded that the interconnection of this information provides a more accurate assessment of bone porosity, contributing to a more accurate estimation of the risk of pathological fractures.

The study brings into discussion new perspectives in the management of osteoporosis through the possibility of evaluating the cytoarchitectonics of bone tissue. The approach, that explores possible beneficial side effects of a widely prescribed medication for patients at high risk of osteoporosis and cardiovascular disease, can optimize the treatment of these patients. The accumulation of evidence provided by various methods supporting the idea that statins could have a double therapeutic potential could influence the way chronic diseases are managed31.

Basic research, including this study, may provide a platform for the development of clinical trials. The results evidenced by long-term simvastatin and fenofibrate administration may provide a rationale for clinical evaluation of their osteoprotective effect on the progression of osteoporosis. The innovative methods used, MRI diffusion and relaxometry, may open new clinical perspectives allowing a more complex evaluation of bone tissue.

Conclusions

Both, fenofibrate and simvastatin, decrease bone strength, the effect being more significant with the increase of the duration of treatment. When administered, at short-term, fenofibrate increases bone porosity more than simvastatin. When administered in the medium term, fenofibrate, unlike simvastatin, increases not only the bone porosity but also the pores’ connectivity. Long-term administration of both substances causes an increase in bone porosity and pore connectivity, the effect being stronger under treatment with fenofibrate than with simvastatin.

Although the method used is only surrogate measures of microstructure, it complies with the current medical practice of assessing bone strength, the study pursuing the possibility of implementing a more complex, non-radiating method in the clinical screening of osteoporosis.

Methods

An experimental study was carried out on bone tissue models, obtained from 24 adult rats, female Wistar Albinos, aged between 16 and 18 months (corresponding to the perimenopausal period in women, i.e. between 47and 52 years) and with an average weight of 300 g, purchased from the Biobase of the University of Medicine and Pharmacy of Târgu Mureș. It is a simple randomized trial, and the animals were housed in compliance with the species standards within the Biobase of the University of Medicine and Pharmacy of Târgu Mureș, benefiting from specialized medical supervision on a daily basis. The medical interventions were approved by the Ethics and Deontology Commission of the University of Medicine and Pharmacy of Târgu Mureș, according to document no. 29/26.06.2012. All experiments were performed in accordance with the University’s guidelines and regulations. The study is reported in accordance with ARRIVE guidelines.

The laboratory animals were divided into two groups, e.g. the rats treated with simvastatin and the rats treated with fenofibrate. The drugs were administered orally by gavage, daily, until euthanasia. The daily dose was 10 mg/kg for both medicinal products. At 14, 16 and 18 weeks after the start of treatment, three animals in each group were euthanized by intraperitoneal injection with a lethal dose of 8–10 mg/kg of ketamine and xyllin. The left femur was preserved in 10% formaldehyde for measurements.

The drug dosage was determined considering both the potential hepatotoxicity of simvastatin and fenofibrate and the extended duration of administration, in order to minimize adverse effects and ensure animal survival throughout the study period. Compared to previously reported experimental protocols, the doses applied in this study were lower, while the treatment duration was longer, allowing for the assessment of chronic effects under conditions of reduced toxicity. The selected dosing regimen aimed to balance experimental validity with ethical considerations regarding animal welfare.

The study used two quantitative, modern methods for analyzing bone tissue: 1D 1H NMR diffusiometry and 2D T2-T2 exchange 1H NMR relaxometry. The nuclear magnetic resonance (NMR) diffusiometry method is a research technique natively used to explore porous materials. This method uses the water molecules inside the pores as “spy molecules” to probe the geometry of the pores, including their size and distribution. The basic principle is to measure the distribution of the self-diffusion coefficients of the molecules, predominantly of water inside the pores. The technique known as PGSE (Pulsed Gradient Stimulated Echo) involves a sequence of three 90o radiofrequency pulses, which generate a stimulated echo. The amplitude of this stimulated echo is determined by the amplitude of the applied magnetic field gradients. The process involves two gradients (with duration denoted by δ): the first gradient is applied between the first two 90o pulses, thus encoding the positions of the molecules in the porous medium. This is followed by a waiting period, usually denoted with Δ, during which water molecules diffuse freely into the pores or change position between pores. The second gradient (δ), applied after the last 90o pulse, decodes the final positions of the molecules according to their diffusive motion.

This method allows the evaluation and analysis of the size and complexity of microscopic cavities in biological structures including bones. By the second method used, the proximal extremity of the femur was analyzed using the Bruker Minispec mq 20 spectrometer, which operates at a frequency of 20 MHz. The pulse sequence used for coding, diffusion and decoding of the positions of water molecules (fixed in formaldehyde) was of the CPMG(T2) – Mz(conservation) – CPMG(T2) type, with a period in which the magnetization of the sample is conserved in the direction of the static magnetic field (20 ms in this case), allowing molecular exchange. The direct measurement of the CPMG echo train decay was performed during the last CPMG (molecule position decoding) sequence, with the gradual increase in the number of CPMG pulses (position coding). The resulting 2D (Laplace-like) spectrum was obtained by applying the two-dimensional Laplace transform.

The T2- T2 molecular exchange, analyzed by means of two-dimensional (2D) maps, will be illustrated with arrows on each figure, indicating the direction of the molecular exchange. The maps will be interpreted in the following way: the peaks on the main diagonal represent the fluid molecules that keep their position in the same pores, such as in trabecular spaces. The full area of these peaks is directly proportional to the number of molecules that remain in the same pores. In contrast, extra-diagonal peaks indicate the presence of a molecular exchange between different pores. The direction of this exchange is represented by arrows that are read clockwise (inversely trigonometric).

The presence of extra-diagonal peaks indicates the existence of communication pathways between different types of pores. The larger the area of these extra-diagonal peaks, the greater the amount of fluid that communicates between pores of different sizes, reflecting the molecular exchange between various hierarchical levels of pores.