Microcomputed tomography of the femur of diabetic rats: alterations of trabecular and cortical bone microarchitecture and vasculature—a feasibility study

Type 2 diabetes mellitus (T2DM) is a disease whose worldwide incidence is increasing [1]. Morbidity and mortality of this disease are mainly due to vascular complications, but more recently, increased risk of fractures has emerged as a serious complication in patients with long-standing or poorly controlled disease [2, 3]. This increased risk of fractures has a distinct propensity for the proximal end of the femur [4‐7] and vertebrae [8] and is problematic because patients with T2DM exhibit compromised bone fracture healing and poorer outcomes after fracture [9‐11].

A paradox of this clinical syndrome is that increased risk of bone fracture is evident despite higher body weight and normal-to-high areal bone mineral density [12], which are generally associated with reduced bone fracture risk. The increased risk of bone fracture is also not explained by a higher frequency of falls among T2DM patients [13]. Several studies using high-resolution peripheral quantitative computed tomography in T2DM patients found that the microarchitecture of the trabecular bone tissue had been preserved but cortical porosity had increased and bone quality was impaired [14‐16]. Definitive elucidation of the pathogenesis of these abnormalities will require further studies.

The use of animal models is essential in research pertaining to T2DM. Zucker diabetic fatty (ZDF) rats constitute a well-documented model for T2DM because these animals have a mutation that is responsible for leptin receptor deficiency and develop insulin-resistant hyperglycaemia and metabolic syndrome at the age of 9 weeks [17]. The ZDF model was used to demonstrate a significant association between T2DM and decreased BMD, trabecular bone volume (Tb.BV), and cortical thickness (Ct.Th) in the vertebrae and in the distal metaphyses of femurs [18‐21]. The ZDF model was also used to test several hypotheses, such as an increase in non-enzymatic glycation [15] [22‐24] and reduction in bone turnover [25‐27], regarding the impaired bone quality observed in T2DM. However, to the best of our knowledge, there is no data available in the literature on the proximal femur, which is a determinant location for the fragility fractures, or on cortical porosity, which is a determinant factor for bone strength. Finally, although microangiopathy is involved in most T2DM complications, bone vascularisation and its potential impaction with bone microstructure remains poorly documented.

The 24-week-old ZDF and ZL rats had an average weight of 439.4 ± 16.35 g and 387.2 ± 10.35 g, respectively. The ZDF rats had higher blood serum glucose levels than the ZL rats (5.88 ± 0.17 versus 2.33 ± 0.15 (p < 0.001)). In addition, the ZDF rats also had significantly increased levels of fructosamine (p = 0.001), alkaline phosphatase (p = 0.005), and calcium (p = 0.001).

The objective of our study was to use micro-CT to compare trabecular and cortical bone microarchitecture and bone vasculature of the femur of ZDF 24-week rats as a model of long standing diabetic conditions to that of their ZL littermates as controls. The ZDF rats are an established model of T2DM which has been used to examine musculoskeletal complications such as bone fragility [18]. Micro-CT enables a separate evaluation of the cortical and trabecular bone compartments; it has also demonstrated its usefulness in the study of bone vascularisation in healthy rats [30‐33].

We observed a significantly lower vBMD in the trabecular compartment of the distal metaphysis of the ZDF rat femurs. Other studies using ZDF rats [20, 21] reported that vBMD varies non-linearly with respect to anatomical location and that the distal femoral metaphysis was the region with the largest vBMD reduction. vBMD, as measured by micro-CT, is an apparent mineral density; it reflects both the amount of bone (bone volume fraction) and the degree of bone mineralisation. Since the degree of bone tissue mineralisation changes very little, vBMD changes mainly reflect changes in the bone volume fraction. Our significantly lower vBMD in the distal diaphysis was clearly related to a significantly lower trabecular bone volume and trabecular number.

Indeed, our study showed a significantly lower trabecular bone volume in the femurs of ZDF rats compared with those of ZL rats; this was observed in the three VOIs located into the head, neck, and distal metaphysis. This finding may be related to the significant differences observed in trabecular number (lower) and spacing (higher) in the ZDF rats compared to the ZL rats. The trabecular thickness was significantly lower only in the femur head of ZDF compared with ZL rats.

The observed lower trabecular bone volume is consistent with the results in literature reports of studies on the vertebrae [19, 20] and distal metaphysis of the femur [18]. In those studies, the differences observed in trabecular bone volume were explained by the number and thickness of trabeculae [17, 18] or thickness only [19]. These studies were carried out at resolutions of 12 and 20 μm on ZDF and ZL 9- and 33-week-old rats. Our study used 6 μm of resolution and 24-week-old rats. Better resolution should improve the determination of any existing differences in trabecular thickness between ZDF and ZL rats. Other studies which used ZDF rats of various ages may be needed in order to determine whether or not there is a difference.

Our differences in SMI in ZDF rats that reflect changes in the microarchitecture of trabecular bone, and may be responsible for fragility and increased fracture risk, are consistent with reports in the literature [17‐19]. The papers in the literature also reported an increase in the connectivity index, indicating an anarchic reorganisation of the bony trabeculae, a result which was not observed in our study.

There is a major need to non-invasively monitor and evaluate the status of cortical bone, which plays a critical role in bone strength [14]. In this respect, mechanical testing of excised femoral necks from human cadavers has showed that the cortex contributes from 40 to 60% of the overall femoral strength [32]. Also, finite element modeling has suggested that, in the human femoral neck region, cortical bone supports 50% of the stresses associated with normal gait [33]. Moreover, administration of some newly developed antiosteoporotic drugs proved especially effective on cortical bone [34]. The mechanical properties of cortical bone depend on several structural and microstructural parameters (such as bone mineral density, cortical thickness, and cortical porosity), which can be assessed using micro-CT with appropriate spatial resolution.

Our study provided evidence of a significantly lower cortical area and of a significant increase in the cortical porosity of the neck and shaft in ZDF rats. These results are in agreement with those reported for the human femoral shaft in one study [19]. However, to the best of our knowledge, cortical porosity has never been studied in ZDF rats before.

Investigation of the bone microvasculature using micro-CT is challenging because vascular opacification is required for the three-dimensional study of the vascular tree [35]. Our study used information from published studies on the topic [28‐30] to establish a protocol involving the use of Microfil® as a contrast agent. Our procedure was complex. We judged injections as satisfactory in only 7 rats out of 12. Such outcomes are consistent with the few reported studies that also obtained unsatisfactory opacification of small blood vessels [29]. Obtaining satisfactory Microfil® injections was not the only challenge. Indeed, the CT densities of bone and opacified vessels are very similar for the two tissues and cannot be distinguished on histograms. Consequently, decalcification of the bone is mandatory. Other options were discussed in the literature. Micro-CT using synchrotron radiation offers both higher spatial resolution and higher contrast and thus provides differentiation of bone tissue from blood vessels without specimen decalcification [28] but is of limited availability. Some studies reported the use of barium sulphate for opacification of blood vessels and, thus, differentiation from bone tissue [31, 33]. Analysis of periosteal vascularisation without decalcification, using a segmentation technique, has also been reported [36] but was not appropriate for our study. Our study demonstrated that bone vascularisation in the ZDF rats was poor compared to that of the ZL rats. The number and percentage of blood vessels in the neck and distal metaphysis of the ZDF rat femurs was lower. Two femoral heads among the ZDF rats tested were unfortunately broken during manipulation, preventing measurements of the vascularisation parameters in the region of interest; in fact, the vascularisation in ZDF rats was poorer than that observed in the ZL rats. The observed difference in the vascular tissue volume may be explained by the significant difference in the number of blood vessels.

To our knowledge, there is no study regarding the bone vascular network and its correlation with alterations in bone quality in the long bones of T2DM rats. Our study applied micro-CT analysis to study bone vasculature and showed impaired bone vascularisation in ZDF rats. It should be noted that the observed vascular changes were more pronounced at the distal metaphysis of the femur where major bone quantity and quality changes were observed. The underlying mechanisms for these events remain unclear. Several hypotheses such as the role of vascularisation in bone remodelling units through complex molecular communications and recruitment of bone cells from pericytes [30], or vasomotor alterations of the principal nutrient artery of the femur from ZDF rats [37], have been proposed. It is therefore possible that the microangiopathy may be at least in part responsible for the bone microarchitecture changes observed in diabetic subjects.

The present study has limitations. First, the sample size dedicated to the study of bone vessels in the femur of rats using micro-CT was quite small. Indeed, we obtained satisfactory opacification of the vessels in only seven out of 12 rats. Second, our analysis was conducted using a resolution of 6 μm, so that an accurate identification of pores was difficult as regards to pore size which varies between 5 and 0.1 mm. The use of a better resolution would probably allow for more accurate analysis of the porosity of bone in ZDF rats. Anyway, the results observed in our study are consistent with literature reports of an increased in porosity in men with T2DM [14, 38, 25].

In summary, our study using micro-CT provided evidence that, firstly, trabecular and cortical bone quantity and microarchitecture were altered in the femur of ZDF rats; secondly, bone vasculature was altered in the femur of ZDF rats; and, thirdly, the main region of interest for studying architecture and vasculature changes in the femur of ZDF rats was the distal metaphysis. Our preliminary study regarding bone vascularisation needs confirmation with larger populations.