Issue 40

I. Doulamis et alii, Frattura ed Integrità Strutturale, 40 (2017) 85-94; DOI: 10.3221/IGF-ESIS.40.08 87 Total serum cholesterol (T-CHOL), high-density lipoprotein cholesterol (HDL-C) serum triglycerides (TG) and serum glucose concentrations were determined enzymatically with commercially available kits (Biosis Biotechnological Applications, Athens, Greece). Due to the nonconfirmed validity of the Friedewald formula for the calculation of low-density lipoprotein (LDL) in rodents this parameter was not included in our study. Moreover, serum adiponectin levels (ADIPO) were estimated with enzyme-linked immunosorbent assay (Mouse Adiponectin ELISA Kit, Intra-assay CV 5.3%, Inter-assay CV 9.9% ABCAM, Cambridge CB4 0FL UK). Mechanical testing After euthanasia, both left and right femur of each animal were resected and stored in gauze immersed into N/S 0.9%. The mechanical behaviour and the strength of the specimens was assessed from three point bending tests. All biomechanical tests were performed within four hours from the time the samples were harvested. Mechanical testing was performed with the use of an electromechanical uniaxial load frame (INSTRON) which was equipped with a high accuracy tension-only load cell (50 N, INSTRON). Because of the use of a tension-only load cell, a custom device had to be used to transform tensional loading to three point bending (Fig. 1). This device comprises two main parts: Part A which was fixed to the load frame's base and B which was attached to its movable crosshead. Part A in- cluded the centrally placed cylindrical pin while Part B included the two support pins (Fig. 1). The diameter of all three pins was 2 mm and the distance between the two support pins was 14 mm. To improve the reliability of the testing pro- cedure the distance between the stationary central pin and the movable support pins was directly measured using a lased micrometer (Keyence LS-3000) (Fig. 1). All samples were loaded with a displacement rate of 5 mm/min until failure. The sampling frequency for the distance between the central and the support pins as well as for the force was 3 Hz. Figure 1 : The custom device that was used to perform three point bending tests with a tension only load cell. The device comprises two parts: (A) which was fixed to the load frame's base and (B) which was attached to its movable crosshead. Before testing, the maximum and minimum external thickness of each sample was measured at the central part of their diaphysis using a digital calliper. Assuming that the cross-section of the diaphysis is elliptical means that the aforementioned maximum and minimum external thickness correspond to the major axis (a) and minor axis (b) of the ellipse respectively (Fig. 2). After the end of the test the actual thickness of the bone cross-section was also measured on the surface of fract- ure. The measurement of wall thickness was repeated four times for each sample: two at opposite sides of the sample’s major axis (t 1 ,t 2 in Fig. 2) and two at opposite sides of the sample's minor axis (t 3 ,t 4 in Fig. 2). In the end, these four measure- ments were used to calculate the average thickness of the sample (t) on the surface of failure. The recorded data in terms of force were used to find the maximum force that was sustained by each sample, namely the fracture force. The force data combined with the measurements of the distance between the central and support pins were used to draw the force/deflection curve of each test and calculate the stiffness of each sample and also their energy to