Rev Osteoporos Metab Miner. 2011; 3 (2): 95-100
Ruiz Mambrilla M5, Domingo Andrés M1, Primo Martín D1
2 Hospital Universitario Marqués de Valdecilla – Universidad de Cantabria – Reticef – Santander
3 Complexo Hospitalario Universitario de Santiago de Compostela (CHUS) – Reticef – Santiago de Compostela
4 Trabeculae® Empresa de Base Tecnológica, S.L. – Ourense
5 Centro Médico de Rehabilitación y Lenguaje – Valladolid
Background: The effects of type 2 diabetes on the microstructure and mass of bone are not clearly defined. The objective of this study has been to assess the microstructural properties and volumetric bone mineral density of Goto-Kazizaki rats, the rat model for non-overweight type 2 diabetes which tries to circumvent the influence of obesity on bone mass.
Material and methods: An experimental study was designed using Goto-Kazizaki rats compared with a control group of non-diabetic Wistar rats of similar weight and with normal glycemia, with densitometric and microstructural studies being carried out on the distal region of the femur using computerised X-ray microtomography (micro-CT).
Results: In the volumetric densitometry no significant differences were found between the two groups. The microstructural study showed that the BV/TV and trabecular connectivity were reduced in the diabetic rats, while the tube-like trabeculae increased to the detriment of plaque-like trabeculae.
Conclusion: The deterioration trabecular bone quality could explain the decrease in biomechanical bone resistance in type 2 diabetes.
Skeletal disorders in diabetes are different according to whether one is dealing with type 1 or type 2 diabetes. Those patients with type 1 diabetes have a decrease in bone mass which implies an increased risk of fracture1,2, whilst type 2 diabetes may present bone mass which is increased, reduced or within normal limits3,4. However, the risk of fracture in type 2 diabetes is increased5,6. This fact could be due to causes both inside, and outside, the bone (retinopathy, neuropathy, drugs, etc) which determine a higher incidence of falls. One fact common in type 2 diabetics is overweight, which constitutes a confusion factor due to the fact that weight is a determining factor of bone mass. Obese people normally have raised bone mineral density. To avoid the influence of this confusion factor an experimental study was designed with Goto-Kakizaki (GK) rats, a substrain of non-obese Wistar rats which develop type 2 diabetes. GK rats have a light to moderate type 2 diabetes which occurs after birth, and develop chronic complications of the disease such as neuropathy and nephropathy7.
The main objective of this study was the assessment using computerised micro-tomography of the densitometry and the trabecular and cortical microstructural properties of GK rats, and to compare them with a group of Wistar rats used as a control, with the intention of evaluating, and in their case, defining, the changes which in these variables induce obesity.
Material and methods
An experimental study was carried out with 4 male GK rats as against a control group of 4 male non-diabetic Wistar rats of a similar weight and with normal glycemia (Taconic Farms Inc. Lille Skensved, Denmark), due to the fact that the Goto-Kakizaki substrain was developed from the Wistar rats. The treatment of the animals and all the experiments were carried out in accordance with Law 14/2007 and with Royal Decree 1201/2005, and following the guidelines of the UNE-EN 30993-3:1994 rules and ISO 10993-2:2006. The rats were fed with a standard diet and had free access to water, did not received any drug treatment, and were sacrificed at 12 weeks in a chamber with CO2.
Microstructural analysis of the bone using micro-CT
Once the animals were sacrificed, the right femurs were extracted in order to carry out the structural analysis. After the extraction, the samples were wrapped in gauze soaked in saline solution and conserved at -20°C until the last moment before analysis. The microstructural analysis of the samples was carried out using computerised X-ray microtomography (micro-CT), using the commercial equipment SkyScan 1172 (SkyScan NV, Aarstelaar, Belgium) in the Trabeculae® research laboratory, Empresa de Vase Technologia, S.L (Ourense, Spain).
The distal region of each femur was scanned with an X-ray source of 50 KV and an intensity of 200 µA, using a voxel size of 8.95 µm. An aluminium filter of 0.5 mm in thickness was positioned to reduce beam hardening artefacts. The rotation step used was 0.4° up to a total of 180° and the exposure time was 1250 ms.
The images obtained were reconstructed using the modified Feldkamp algorithm8, using the NRecon 1.5 software application (SkyScan NV, Aarstelaar, Belgium). The transverse sections resulting from the reconstruction stage were used for the quantitative analysis of the trabecular and cortical bone microstructure, using the application CTAn 22.214.171.124 (SkyScan NV, Aarstelaar, Belgium), after their segmentation into binary images using locally adapted thresholding9.
For the analysis of the trabecular bone a metaphyseal-diaphyseal region of interest (cortical bone excluded) of 2.5 mm was selected, starting at a distance of 1.0 mm from the growth plate in a proximal direction. For the analysis of the microstructural properties of the cortical bone a region of interest of 1.0 mm was taken, starting at 4.0 mm from the growth plate (Figure 1).
The quantitative variables which were determined for the trabecular region were: volumetric bone fraction (BV/TV), specific bone surface (BS/BV), bone surface density (BS/TV), trabecular thickness (Tb.Th), trabecular separation (Tb. Sp), trabecular number (Tb.N), trabecular pattern factor (Tb.Pf), structural model index (SMI), and degree of anisotropy (DA). The different variables were measured directly using methods described in the literature10,11. The non-metric variables, SMI and Tb.Pf, were calculated directly from the three dimensional model. The SMI indicates the relative prevalence of plate-like or rod-like trabeculae, with a higher presence of plates being indicated the nearer its value is to zero12. For its part, Tb.Pf is an inverse index of connectivity, measured from the calculation of the relative convexity or concavity of the bone surface13. A higher value of Tb.Pf the trabecular network shows a poorer connectivity, which also implies a reduction in mechanical resistance. The DA is a measure of the alignment of the trabeculae in a determined direction, calculated in such a way that 0 is complete isotropy and 1 is complete anisotropy.
In the case of the cortical region, the parameters calculated included: cortical thickness (Ct.Th), the average transverse area of the bone (B.Ar), the average polar inertial moment (I) and the eccentricity (Ecc). I is a basic index of mechanical resistance which indicates the resistance to rotation of a transverse section in a determined axis (assuming uniform biomechanical properties). Ecc is a parameter which indicates the difference in elongation of a transverse section with respect to a circular form (a circle is considered to be an ellipse with zero eccentricity).
Determination of volumetric bone mineral density
Using the images obtained though micro-CT, the volumetric bone mineral density (BMDv) was determined, both in the cortical and trabecular regions. Direct calibration was used with attenuation coefficients of calcium hydroxyapatite models of known density (250 and 750 mg/cm3). The method of calculating the BMDv differed slightly in different areas of the bone, since in the case of the trabecular region it refers to a volume of bone and medullar tissue, whilst in the case of the cortical region it was limited to a volume occupied solely by calcified lamellar bone.
The data obtained were put into a text database which was exported to the statistical software package IBM SPSS Statistics 19 (IBM Corporation, Somers, NY, US) for their subsequent statistical analysis. The individual results were reviewed in order to avoid loss of data and unusual values. Then the descriptive analysis of the variables of the study was proceeded with. The descriptive statistics of the numerical variables were expressed as an average ± standard deviation, maximum value and minimum value.
The comparative statistical study of the numerical data was carried out by means of an single factor variance analysis (ANOVA) and the Tukey HSD test for multiple comparatives. In those cases which did not comply with homogeneity of variance criteria, the Broen-Forsythe test was applied for the variance analysis and the Games-Howell test for the multiple comparatives.
The level of statistical significance was established at values of p<0.05 for all the variables analysed.
The GK rats had a weight of 385 ± 23 g and glycemia of 195 ± 84 mg/dl, which confirms the presence of diabetes in this group, while the Wistar rats had a weight of 395 ± 35 g and glycemia of 124 ± 15 mg/dL (p<0.01). The results of the bone mineral density volumetry are reflected in Table 1. BMDv did not show differences between the two groups, neither in the cortical section, nor the trabecular. Although there appears to be a loss of trabecular bone mass in the diabetic rats, this did not reach significant levels, probably due to the sample size used, or the development time of the diabetes.
Table 2 shows the results of the microstructural variables in the trabecular region. Statistically significant differences were observed in the volumetric bone fraction (BV/TV), indicating a loss of trabecular bone in the diabetic rats as compared with the control group. The considerable increase in Tb.Pf in the GK rats confirms, also, a significant loss of trabecular connectivity in diabetes. On the other hand, the increase in SMI shows a prevalence of tubular trabeculae in diabetic rats in comparison with the control group, in which plate-like trabeculae predominate. Although the BS/TV, the Tb.Th and the Tb.N are reduced in diabetic rats, at the same time the Tb.Sp is increased, together indicating a deterioration in the trabecular microarchitecture (Figure 2), their values not reaching statistically significant levels.
In the cortical region, although the diabetic group appears to have a reduction in the thickness of the cortical wall, this being determined through the transverse sections (Cs.Th) or assumed in the three-dimensional model (Ct.Th), this does not reach statistically significant levels (Table 3). The variables B.Ar, I and Ecc show very similar values in the two groups. The cortical regions of two representative samples can be seen in Figure 3.
The study carried out shows that diabetic rats have a volumetric bone density similar to non-diabetic rats. However, differences are observed in the trabecular bone in the structural parameters. There is a reduction in BV/TV, a lower trabecular connectivity and a predominance of cylindrical trabeculae in the GK rats. The trabecular structural variables in which there are significant differences between the diabetic and control rats are those related to bone resistance. The lower quantity of bone indicated by the reduction in BV/TV, and the great loss of trabecular connectivity revealed by the increase in Tb.TPf in the diabetic group, result in an evident reduction in biomechanical resistance. In addition, it has been shown that the tube-like trabeculae, which predominate in the diabetic group, are less resistant to mechanical load than the plate-like trabeculae12, more abundant in the control group, from which is deduced the SMI value. All these data indicate that, although their bone density is normal, the GK rats would have a lower biomechanical resistance, which could suggest that the higher prevalence of fractures which occurs in type 2 diabetes would be related to alterations in bone quality.
There are few studies carried out with this experimental model. Zhang et al. measured, in a group of GK rats, the bone mineral density using DXA and the microstructure with classic histomorphometric techniques in 2D7. These authors found a reduction in bone mass and a change in the histomorphometry with a decrease in BV/TV, similar to that found in our study. Ahmad et al., using peripheral quantitative computerised tomography (pQCT), observed a decrease in volumetric bone mineral density predominantly in the trabecular section14. The measurements were taken in the humerus, tibia and metatarsals of female rats, which could explain the differences observed from our results. In none of the regions analysed in this work were there found differences in the cortex. However, another work, using radiogrametric techniques in a group of 10 GK rats observed a decrease in cortical thickness in the metatarsal and humerus15. The data observed are heterogeneous, probably due to the different techniques used and the different places where measurements were taken. However, in all of them, trabecular affectation predominates and, when it was determined, a reduction in biomechanical bone resistance. Another model of non-obese diabetic rat is that of Zucker rats, developing the disease progressively until the serious complications of the disease appear. These rats show a decrease in bone mass, both cortical and trabecular, smaller sized large bones, and a deterioration in the biomechanical and microstructural properties of the cortical and trabecular bone16,17.
Various mechanisms may explain this change. Glucose represents the principal source of energy to the osteoclasts, with hyperglycemia being responsible for an increase in osteoclast activity, with an increase in bone remodelling and a decrease in the quantity and quality of bone2. On the other hand, hyperglycemia provokes the non-enzymatic glycosylation of the bone proteins, damaging bone quality18. In turn, glycosuria increases hypercalciuria with changes to the PTH/vitamin D system. These deleterious effects on bone quality may be partially compensated for by an increase in bone mass associated with obesity19. An alteration in calcium metabolism may also contribute to the deterioration of bone quality. In diabetics an increase in calciuria has been described, which has been related to hyperglycemia and glycosuria. This provokes secondary hyperparathyroidism which exerts a prejudicial effect on the bone, especially on the trabecular section20. The alteration of the vitamin D and parathormone metabolism is particularly prominent in patients with reduced renal function. Microangiopathy can alter endothelial function21, and macroangiopathy with arteriosclerosis can cause a reduction in blood supply to the bone22,23. On the other hand, in patients with neuropathy a change in the load on the bone can also contribute to the loss of bone mass. We can say, therefore, that there are multiple mechanisms which exert a deleterious effect on bone in experimental animals with type 2 diabetes which can explain the alteration in bone quality in these models.
In conclusion, we can say that Goto-Kakizaki rats are a valid model for the study of type 2 diabetes, since they eliminate a significant confusion factor, overweight. Although the sample size is small, we found a deterioration in the microstructure in the femoral trabeculae, with, however, the volumetric bone mineral density being preserved.
1. Olmos JM, Pérez Castrillón JL, García MT, Garrido JC, Amado JA, González Macías J. Bone densitometry and biochemical bone remodeling markers in type 1 diabetes mellitus. Bone Miner 1994;26:1-8.
2. Carnevale V, Romagnoli E, D’Erasmo E. Skeletal involvement in patients with diabetes mellitus. Diabetes Metab Res Rev 2004;20:196-204.
3. Cortés Sancho R, Pérez Castrillón JL, Martín Escudero JC, Iglesias S, Álvarez Manzanares P, Ramos R. Type 2 diabetes mellitus as a risk factor for hip fracture. J Am Geriatr Soc 2004;52:1778-9.
4. De Liefde II, Van der KM, De Laet CE, Van Daele PL, Hofman A, Pols HA. Bone mineral density and fracture risk in type-2 diabetes mellitus: The Rotterdam Study. Osteoporos Int 2005;16:1713-20.
5. Vestergaard P, Rejnmark L, Mosekilde L. Relative fracture risk in patients with diabetes mellitus, and the impact of insulin and oral antidiabetic medication on relative fracture risk. Diabetologia 2005;48:1292-9.
6. Janghorbeni M, Van Dam RM, Willet L, Hu FB. Systematic review of type 1 and type 2 diabetes mellitus and risk of fracture. Am J Epidemiol 2007;166:495-505.
7. Zhang L, Liu Y, Wang D, Zhao X, Qiu Z, Ji H, Rong H. Bone biomechanical and histomorphometrical investment in type 2 diabetic Goto-Kakizaki rats. Acta Diabetol 2009;46:119-26.
8. Feldkamp LA, Davis LC, Kress JW. Practical cone-beam algorithm. J Opt Soc Am 1984;A6:612-19.
9. Xuan L, Sasov A. Cluster reconstruction strategies for micro CT and nano Ct scanners. In: Proceedings of the Fully Three-Dimensional Image Reconstruction Meeting in Radiology and Nuclear Medicine, July 6-9, 2005, Salt Lake City, UT, USA.
10. Hildebrand T, Rüegsegger P. A new method for the model independent assessment of thickness in three-dimensional images. J Microsc 1997;185:67-75.
11. Ulrich D, van Rietbergen B, Laib A, Rüegsegger P. The ability of three dimensional structural indices to reflect mechanical aspects of trabecular bone. Bone 1999;25:55-60.
12. Hildebrand T, Rüegsegger P. Quantification of bone microarchitecture with the structure model index. Comput Methods Biomech Biomed Eng 1997;1:15-23.
13. Hahn M, Vogel M, Pompesius-Kempa M, Delling G. Trabecular bone pattern factor: A new parameter for simple quantification of bone microarchitecture. Bone 1992;13:327-30.
14. Ahmad T, Ohlsson C, Sääf M, Östenson CG, Kreicbergs A. Skeletal changes in type-2 diabetic Goto-Kakizaki rats. J Endocrinol 2003;178:111-6.
15. Östenson CG, Fière V, Ahmed M, Lindström P, Brismar K, Brismar T, et al. Decreased cortical bone thickness in spontaneously non-insulin-dependent diabetic GK rats. J Diabetes Complications 1997;6:319-22.
16. Prisby RD, Swift JM, Bloomfield SA, Hogan HA, Delp MD. Altered bone mass, geometry and mechanical properties during the development and progression of type 2 diabetes in the Zucker diabetic fatty rat. J Endocrinol 2008;199:379-88.
17. Reinwald S, Peterson RG, Allen MR, Burr DB. Skeletal changes associated with the onset of type diabetes in the ZDF and ZDSD rodent models. Am J Physiol Endocrinol Metab 2009;296:E765-74.
18. Yamagishi S, Nakamura K, Inoue H. Possible participation of advanced glycation end products in the pathogenesis of osteoporosis in diabetic patients. Med Hypotheses 2005;65:1013-5.
19. Pérez Castrillón JL, De Luis D, Martín Escudero JC, Asensio T, Del Amo R, Izaola O. Non-insulin-dependent diabetes, bone mineral density, and cardiovascular risk factors. J Diabetes Complications 2004;18:317-21.
20. Hofbauer LC, Brueck C, Singh SK, Dobnig H. Osteoporosis in patients with diabetes mellitus. J Bone Miner Res 2007;22:1317-28.
21. Sanada M, Taguchi A, Higashi Y, Tsuda M, Kodama I, Yoshizumi M, et al. Forearm endothelial function and bone mineral loss in postmenopausal women. Atherosclerosis 2004;176:387-92.
22. McNair P, Christensen MS, Christiansen C, Madsbad S, Transbøl I. Is diabetic osteoporosis due to microangiopathy? Lancet 1981;1(8232):1271.
23. Vogt MT, Cauley JA, Kuller LH, Nevitt MC. Bone mineral density and blood flow to the lower extremities: The study of osteoporotic fractures. J Bone Miner Res 1997;12:283-9.