4.3. Analyses
4.3.1. Bone resorption rate
Four-week old rats were injected subcutaneously with 1 ml of a solution containing 5Ci/ml (185 GBq/L) of [7-3H(N)];-tetracycline (NEN Life Science Products, Boston, MA) dissolved in distilled water. The injections were repeated weekly for 5 weeks. One week after the last [3H];tetracycline injection, the rats were housed in individual metabolic cages for a 24-hour baseline urine collection. After that, urine was collected twice a week for 31 days, and in study III, additionally once a month for two months. The urine collection period was always 24 hours, so as to avoid possible confusion caused by the diurnal rhythm (Mühlbauer and Fleisch 1990).
The volume of urine excreted was measured, and the amount of 3H radioactivity present in a 1-ml aliquot was determined with a 1215 Rackbeta II scintillation counter (Wallac Co., Turku, Finland), using Hydrofluor (National Diagnostics, Manville, NJ) as the liquid scintillation counting solution. The total excretion of 3H was calculated as an indicator of the amount of resorbed bone mineral as described by Klein and Jackman (1976).
After the urine collection period, the rats were killed, and their tibiae and scapulae were prepared for the determination of the 3H content of bone. The total 3H radioactivity per whole bone was determined using a modification of the method described by Klein and Jackman (1976). The tibial epiphyses and the bone marrow were carefully removed. After drying at 60 C for 24 hours, the bones were weighed and pulverized with a micromill Mixer Type III 695 (Retsch, Haan, Germany). Thereafter, 25 mg of the pulverized bone was suspended in a sealed tube in a 3:1 mixture of concentrated HCl (pro analyse, Riedel-de Haen, Seelze, Germany) and concentrated HNO3 (pro analyse, Merck, Darmstadt, Germany). The 3H radioactivity of the resulting suspension was measured as described above. The total amount of 3H left in the tibiae and the scapulae was calculated.
4.3.2. Analyses of bone inorganic fraction
Whole bone wet weight (with the marrow) of tibia was determined with a weighing machine (Mettler A30; Mettler Instrumente, Zürich, Switzerland), followed by a pycnometric measurement of bone density. Humerus was used for the other analyses. After epiphyses and bone marrow of the humerus had been carefully removed, and samples dried at 60C for 24 h, the bone sample was pulverized with a micromill Mixer Type III 695 (Retsch, Haan, Germany). Bone calcium was determined by atomic absorption spectrophotometry (Perkin Elmer model 2380, Perkin Elmer Corporation, Norwalk, CT), using samples dissolved in a solution of HCl and HNO3 (2:1 conc.). Bone phosphorus was analyzed as described by Fiske & Subbarow (1925). Bone ash weight was determined by ashing 25 mg of the pulverized bone at 900C for 24 h.
4.3.3. Trabecular bone volume
The proximal tibiae were cut sagittally into two equal halves with a diamond saw, dehydrated with ethanol (40%), and embedded in methylmethacrylate as described by Baron et al. (1983). Undecalcified sections of 5m were cut with a Polycut S heavy duty microtome (Reichert-Jung, Leica Instruments GmbH, Nussloch, Germany), and stained according to the von Kossa method (Dickson 1984). Sections were taken near the sagittal midline of the tibia at five levels, 50 m apart, and one microscopic field per section was evaluated. Trabecular bone volume was measured in an area of 5 mm2, at 4x objective magnification, using a computer image analyzer (MCID, Model M1, Imaging Research Inc., Brock University, Ontario, Canada). The area situated within 1 mm from the upper surface of growth plates, as well as all trabeculae in contact with the cortices, were excluded from the measurements.
4.3.4. Analyses of bone organic fraction
Tibia was prepared for the determination of bone collagenous structures in study IV, and humerus in study III. The bones were decalcified at 4 °C with several changes of 0.5 mol/L EDTA, pH 7.2 for 5 weeks. After washing with 0.9 % NaCl, the bones were lyophilized, and pulverized with the micromill. The bone samples were weighed, freeze-dried, and hydrolyzed in 6 mol/L HCl at 110 °C for 24 hours in sealed tubes. A part of the hydrolyzate was subjected to partition chromatography for the analysis of pyridinium crosslinks according to the method of Black et al. (1988). Reversed phase high-performance liquid chromatography (HPLC) was carried out by the method of Eyre et al. (1984), with the modifications of Palokangas et al. (1992). Hydroxyproline was analyzed by HPLC, following derivatization by means of the o-phthalaldehyde/9-flurenylmethoxycarbonyl chloride system, originally described by Teerlink et al. (1989), and modified by Palokangas et al. (1992). The HPLC analyses were performed using the Merck Hitachi chromatograph, which included a pump, model 655 A-12 Liquid Chromatograph, the L-5000 LC Controller, the AS-4000 Intelligent Auto Sampler, the F-1000 Fluorescence Spectrophotometer and the D-2000 Chromato Integrator. The Ultrashere ODS column (C18, 5 µ, 250 x 4.6 mm) from Beckman, and the sample loops of 50 µl and 100 µl, were used for the analysis of hydroxyproline and pyridinium crosslinks. The flow rate used was 1 ml/min.
4.3.5. Bone biomechanical properties
After preparation, left tibia and both femurs were wrapped in gauzes saturated with physiological saline and stored at -20C until used. The storage at -20C has been shown not to affect bone biomechanical properties (Peng et al. 1994b). Before testing, the bones were thawed at room temperature, and kept moist until the test was completed. To avoid confusion of earlier loadings, all mechanical tests were performed with different bones. The left femur was used for the torsion test, the right femur for the loading test of femoral neck, and the tibia for the three-point bending test.
The three-point bending test, and the loading test of femoral neck were performed using a material testing machine constructed by Timo Jämsä, PhD, at the Technical Services Department of the Medical Faculty, University of Oulu (Jämsä 1998). The testing machine is based on the lever arm principle, as described by Peng et al. (1994b). The interchangeable compression head, mounted on the pressing rod for different tests, transmits compressive force to the specimen moving at a constant speed of 0.155 mm/sec. The compressive force is measured by a sensor attached to the stationary part of the compression stage. A load-deformation curve was recorded during the tests by a plotter (Perkin-Elmer 165, Hitachi Ltd., Japan). Prior to testing, the machine was calibrated using a standard weight.
In the three-point bending test (Figure 4-1), performed as described by Peng et al. (1994b), a supporter with two loading points, 13mm apart from each other, was used on the stage of the testing machine. Lateral surface of the tibia at tibiofibular junction was placed upon the first point, and proximal tibia upon the other. A press head compressed the middle of the tibial shaft until fracture occurred. The press head was rounded to avoid cutting into the bone when loaded.
Stress, strain and Young"s modulus were derived from load-deformation curves obtained, by using equations described by Turner and Burr (1993). Stress, which is defined as force per unit area, was calculated as FLc/4I, where F is applied force (from the load-deformation curve), L is the distance between the loading points, c is the distance between the bone surface and the bone cross-sectional center, and I is the bone cross-sectional moment of inertia. Roundness of tibia cross-sections was within 0.92-0.94 and cross-sectional moment of inertia was determined by the equation of round specimens. Strain, which is defined as percentage change in length, or relative deformation, was calculated as 12cd/L2, where d is displacement (from the load-deformation curve). Young"s modulus, which is defined as the slope of the stress-strain curve within the elastic region, and measures the intrinsic stiffness of the material, was calculated as F/d x L3/48I.
Figure 4-1. Schematic presentation of the mechanical tests of the bones. F is the applied force, T is the applied torque, and the arrows represent the direction of the loading.
In the loading test of femoral neck (Figure 4-1), performed as described by Tuukkanen et al. (1994), the head of the femur was loaded with a force parallel to the shaft of the femur until failure. A thick polymethyl methacrylate plate with several holes of different sizes, and grooves for the third trochanter of the femur, was used as a supporter for the bone. The femur was cut between the middle and lower third of the shaft, and the proximal part was inserted perpendicularly and tightly into a suitable hole until the lesser trochanter of the bone touched the surface of the plate. The concave compressing head used was 2.5 mm in diameter. Area of the femoral neck was measured at the point of fracture and the ratio load/area was calculated.
The torsion test of femur was performed using a method reported by Lepola et al. (1993), with a machine constructed by Timo Jämsä, Ph.D., at the Technical Services Department of the Medical Faculty, University of Oulu (Jämsä 1998). One of the heads of the machine rotates at an angular velocity of 6 per second, and the other head is stationary. The sensors for measuring the torsional load are attached to the stationary head. A load-deformation curve was recorded during the tests by a paper recorder (Goerz RE 511, Austria). Prior to testing, the machine was calibrated against a given torque of 0.5 Nm.
Each end of the femur was placed concentrically in a cavity of a nut (M8, Kanthal AB, Hallstahammar, Sweden) by a standardized method, and fixed with dental stone (Fujirock, G-C Dental Industrial Co., Tokyo, Japan). The femur with the nuts was inserted into two head sleeves of the torsion machine, and torsion was performed by twisting the bone inward.
Shear stress and shear modulus of elasticity were derived from load-deformation curves obtained by using equations described by Turner and Burr (1993). Shear stress was calculated as Tr/J, where T is applied torque (from the load-deformation curve), r is the radius of the bone cross-section, and J is the polar moment of inertia of the bone cross-section, which was determined by using the equation of round specimens. Shear modulus of elasticity was calculated as T/θ x L/K, where T/θ is the slope of the load-deformation curve, L is the length of the unembedded portion of the bone, and K is the torsional constant, which is equal to the polar moment of inertia for circular cross-sections.
Length of the bones was measured with calipers. Cross-sectional views of the bones at the point of fracture were photographed under a microscope. The point of fracture in the three-point bending test was standardized by always placing the bone similarly in the testing machine. In the loading test of femoral neck and in the torsion test, however, the cross-section had to be measured using a standardized position of the opposite bone. This was done by cutting the opposite femur and femoral neck perpendicularly at their narrowest position. The cross-sectional areas and diameters of the bones were measured from the micrographs using Image Measure Computer Program (Microscience, Washington DC).