INTERRELATIONSHIPS BETWEEN DENSITOMETRIC, MORPHOMETRIC, AND MECHANICAL PROPERTIES OF THE TIBIA IN TURKEYS

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1 Bull Vet Inst Pulawy 51, , 2007 INTERRELATIONSHIPS BETWEEN DENSITOMETRIC, MORPHOMETRIC, AND MECHANICAL PROPERTIES OF THE TIBIA IN TURKEYS WITOLD KRUPSKI, AND MARCIN R. TATARA 1 II Department of Radiology, Medical University of Lublin, Lublin, Poland 1 Department of Animal Physiology, Faculty of Veterinary Medicine, Agricultural University of Lublin, Lublin, Poland krupskiw@wp.pl Received for publication October 04, 2007 Abstract The aim of the study was to evaluate the interrelationships between parameters assessed with the use of a computed tomography technique and mechanical properties of the tibia in male turkeys at different stages of skeletal development. Healthy male turkeys (N = 120) were kept under standard rearing conditions and randomly selected from the farm flock at the age of 4, 8, 12, and 20 weeks of life to obtain a right tibia for the investigations. Using a computed tomography technique, volumetric bone mineral density of the trabecular and cortical bone, mean volumetric bone mineral density, total bone volume, and cortical bone area of the tibia were estimated. Geometrical properties, such as cross-sectional area, second moment of inertia, mean relative wall thickness, and cortical index, were derived from computed tomography measurements of horizontal and vertical diameters of the tibia in the midshaft. Using a three-point bending test, the mechanical parameters were estimated in terms of maximum elastic strength and ultimate strength of the tibia. A Pearson s correlation coefficient was determined for all the investigated variables. The obtained results showed age-related changes of the skeletal system in growing turkeys in terms of morphological, densitometric, and mechanical properties. Positive correlations among the investigated parameters were found, except for mean relative wall thickness and cortical index, which were negatively correlated with these parameters. Morphometric and densitometric parameters assessed with the use of computed tomography were significantly correlated with maximum elastic strength and ultimate strength proving the usefulness of this technique for predicting mechanical properties of bones. In conclusion, this study showed computed tomography as a precise and non-invasive technique for the determination of skeletal system properties that may serve to monitor in vivo the dynamics of the changes of bone metabolism in studies on physiological, nutritional, and pharmacological factors influencing the skeletal system. Key words: turkey, tibia, bone mineral density, mechanical endurance, quantitative computed tomography. Bone tissue plays supportive, locomotory, protective, and metabolic functions in vertebrates. All these functions are crucial for an effective production of animals and optimal quality of life in humans, especially in the elderly (8, 10, 17). Mechanical properties of bones depend on many factors, among of which bone geometry, bone quantity, and quality in terms of mineral content, microarchitecture, microdamages, as well as collagen structure and maturation belong to the most important ones (11). During skeletal growth and during aging of the skeleton, bone mechanical endurance is related to constant changes of morphological properties and bone mineral density. An increased bone size and mineral density were reported as essential factors improving the bone s mechanical properties, whereas its decreased values induced opposite effects (6, 12, 14, 15). Bone mineral density determined with the use of dualenergy X-ray absorptiometry (DEXA) method was reported as a good predictor for the vertebral fracture risk (7, 18). Computed tomography (CT), is a highly advanced technique of diagnostic imaging invented by Sir Godfrey Hounsfield in 1972 in Great Britain using the theoretical basis provided by Allan Cormack. The importance of the discovery of this diagnostic technique was underlined by the Nobel Prize in Physiology or Medicine for Hounsfield and Cormack in This method provides highly precise imaging of anatomical structures in both humans and animals with the possibility for analysis of tissues and organs in series of cross-sectional slices. As opposed to X-ray radiographs showing two-dimensional anatomical structures, advanced software of CT scanners enables the conversion of obtained digital data into multiplanar reconstructions (MPR) of the investigated structures and its analysis at any trajectory. Moreover, three dimensional (3D) reconstructions with the use of surface shaded display (SSD) technique or volumetric analysis using volume rendering technique (VRT) of organs may be performed (17). Quantitative computed tomography

2 622 (QCT) is a highly respected and non-invasive method for volumetric bone mineral density determination in any part of the skeleton. In contrast to DEXA method, which enables areal determination of the bone s mineral density (expressed in g/cm 2 ), QCT allows volumetric analysis of trabecular and cortical bone density (expressed in g/cm 3 ), independent of one another. Another advantage of QCT method for the determination of volumetric bone mineral density (vbmd), in comparison with DEXA method is the fact that with the use of this method, vbmd can be easy measured without errors resulting from surrounding soft tissues and possible osteoarthritic changes (6, 9). The aim of this study was to evaluate the interrelationships between parameters assessed with the use of computed tomography technique and mechanical properties of the tibia obtained from male turkeys at different stages of skeletal development, namely 4, 8, 12, and 20 weeks of life. To achieve this goal, Pearson s correlation coefficient was estimated between such variables as bone weight, bone length, vbmd of the trabecular and cortical bone, mean vbmd, total bone volume, cortical bone area, cross-sectional area, second moment of inertia, mean relative wall thickness, maximum elastic strength, and ultimate strength. Material and Methods Experimental design and sampling procedure. The study was performed on 120 healthy T5 male turkeys (Holly Berry Hatcheries Ltd., UK), randomly selected from the farm flock at different stages of their development, and divided into 4 agedifferentiated groups. The first group (N = 30) consisted of turkeys slaughtered at the age of 4 weeks while to the second group belonged 8-week-old animals (N = 30). Turkeys slaughtered at the age of 12 and 20 weeks belonged to the third (N = 30) and fourth (N = 30) groups, respectively. During the course of the study, all the animals were kept under standard rearing conditions with constant access to fresh water and appropriate feed supplied ad libitum in accordance with several stages of the production cycle. Morphological examination of the tibia. Immediately after post-mortem isolation of right tibiae, bone samples were cleaned from remaining soft tissues, their weight and length were measured, and they were frozen at 25ºC until further analyses. Computed tomography evaluation of the tibia. The bone samples were thawed for 2 h at room temperature before being scanned with the use of computed tomography technique in SOMATOM EMOTION SIEMENS apparatus supplied with Somaris/5 VB10B software (Version B10/2004A, Siemens, Germany). All bone samples were scanned using 2-mm thick, sequential, cross-sectional scans. Using quantitative computed tomography method, the volumetric bone mineral density (vbmd) of the trabecular and cortical bone was determined. Trabecular bone (Td) and cortical bone (Cd) mineral densities were determined using 2-mm thick QCT scans placed in the distal metaphysis and at 50% of the tibia length, respectively. While Cd was determined in all groups of turkeys, Td of the tibia was measured in 12 and 20- week-old birds only. Furthermore, the cortical bone area (A Cb ) on cross-section at 50% of tibia length was measured in all the groups of turkeys. Using Volume Evaluation application package (software Version B10/2004), the total bone volume (B vol ) of each tibia was determined automatically. For B vol calculations, the volume-of-interest (VOI) was defined by limiting the minimum and maximum density for the investigated bone at 60 and 3000 Hounsfield units (HU), respectively. Volume Evaluation application package was used also to determine the mean volumetric bone mineral density (MvBMD) for each tibia. Analysis of geometrical properties of the tibia. On the basis of measurements of horizontal and vertical diameters (both internal and external) performed on CT mid-diaphyseal cross-section of the tibia, the geometrical parameters such as cross-sectional area (A), second moment of inertia (Ix), mean relative wall thickness (MRWT), and cortical index were derived (2, 4, 5, 16). Analysis of mechanical properties of the tibia. Mechanical properties of tibia were estimated with the use of the three-point bending test in INSTRON 3369 apparatus (Instron, Canton, USA) linked with a computer registering the relationship between forces perpendicular to the longitudinal axis of the bone and the resulting displacement (4, 5). The distance between bone supports was set at 40% of total tibia length and the crosshead loaded bone samples at the midshaft with constant speed of 20 mm/min. The values of maximum elastic strength (Wy) and ultimate strength (Wf) were obtained. Statistical analysis. Statistical analysis was performed using Statistica software (version 6.0). All the data were presented as means ± SEM. The differences of mean values among the age-differentiated groups were tested for statistical significance with the use of one-way ANOVA and post hoc Duncan s test. Pearson s correlation coefficient was determined between all the investigated variables. Differences showing P<0.05 were considered statistically significant. Results Turkeys at the age of 4, 8, 12, and 20 weeks of life reached body weight values of 1.17 ± 0.01 kg, 4.08 ± 0.11 kg, 8.53 ± 0.17 kg, and ± 0.32 kg, respectively. Statistically significant differences of body weight values were found among all the investigated groups of turkeys (P<0.001). The results of morphometric, densitometric, and mechanical analyses of the tibia are shown in Table 1. The lowest values of bone length, bone weight, trabecular bone mineral density, cortical bone mineral density, mean volumetric bone mineral density, total bone volume, cortical bone area, cross-sectional area, second moment of inertia, maximum elastic strength, and ultimate strength were in 4-week-old turkeys, followed by the groups of 8 and 12-week-old birds.

3 623 Table 1 Morphometric, densitometric, and mechanical properties of the tibia of turkeys at different stages of their development Parameter Age of turkeys 4 weeks 8 weeks 12 weeks 20 weeks Bone weight (g) 10.6 a ± b ± c ± d ± 2.0 Bone length (mm) a ± b ± c ± d ± 1.2 Trabecular bone mineral density (g/cm 3 ) a ± b ± Cortical bone mineral density (g/cm 3 ) a ± b ± b ± c ± Mean volumetric bone mineral density (g/cm 3 ) a ± b ± c ± d ± Total bone volume (cm 3 ) 6.3 a ± b ± c ± d ± 1.1 Cortical bone area (mm 2 ) 14.3 a ± b ± c ± d ± 1.3 Cross-sectional area (mm 2 ) 16.7 a ± b ± c ± d ± 1.3 Second moment of inertia (mm 4 ) 41.3 a ± b ± c ± d ± 67.3 Mean relative wall thickness a ± b ± c ± d ± Cortical index 37.6 a ± b ± c ± d ± 0.4 Maximum elastic strength (N) 106 a ± b ± c ± d ± 24 Ultimate strength (N) 193 a ± b ± c ± d ± 28 abcd Values within a row that do not share common superscript letter differ significantly for P<0.05. The highest values of these parameters were obtained in 20-week-old turkeys. The lowest values of mean relative wall thickness and cortical index were found in 20-week-old turkeys, followed by the groups of 12- and 8-week-old turkeys. The highest values of MRWT and CI were found in turkeys at the age of 4 weeks (Table 1). The values of Pearson s correlation coefficient between all the investigated parameters are shown in Table 2. Significantly positive correlations were found between such parameters as bone weight, bone length, Td, Cd, MvBMD, B vol, A Cb, A, Ix, Wy, and Wf. A positive correlation was also found between MRWT and CI (r = 0.982); however, both these parameters were negatively correlated with all the other investigated parameters (Table 2). Discussion The growth and development of the skeletal system in mammals and birds until the achievement of peak bone mass (PBM) is characterised by changes in bone length, bone geometry, and bone mineral density. The Bone s length, geometrical parameters, and bone mineral density, increase with the age until the end of the skeletal maturation process when the maximum amount of bone tissue is reached. After this event, the bone mass of the skeleton results from the amount of bone acquired at skeletal maturity and age-related bone loss (12, 13). In the current study, significant changes of the investigated parameters of the tibia among the agedifferentiated groups of turkeys were demonstrated in accordance with body weight gain. Bone weight, bone length, mean volumetric bone mineral density, total bone volume, cortical bone area, cross-sectional area, and second moment of inertia were significantly different at each of the investigated stages of skeletal development of the turkeys and a gradual increase in these values with the age was observed. Similar changes were observed when volumetric bone mineral density of the trabecular and cortical bone was analysed. As the consequence of age-related increase in bone size, mainly in external dimensions of the tibia, the values of mean relative wall thickness and cortical index showed regular decrease in accordance with the increasing age of the turkeys. All these age-related changes of the morphometric and densitometric properties of the tibia resulted in gradually increasing values of the mechanical parameters, such as maximum elastic strength and ultimate strength. The obtained results also showed interrelationships between morphometric, densitometric, and mechanical properties of the tibia in the turkeys. Positive correlations were found among bone weight, bone length, total bone volume, cortical bone area, cross-sectional area, second moment of inertia and mechanical parameters of the tibia, such as maximum elastic strength and ultimate strength, clearly indicate that developmental changes of the skeletal system, which increase in all these morphometrical parameters, improve mechanical endurance of bones to withstand higher skeletal loading related to body weight gain. Positive correlations of trabecular bone mineral density, cortical bone mineral density, and mean volumetric bone mineral density with maximum elastic strength and ultimate strength of the tibia show significant contribution of bone mineralisation to mechanical properties of bones. The fact that MvBMD and Cd seem to be better predictors of bone mechanical properties than Td is noteworthy. This fact may be explained by different metabolic activity and functions of both these bone compartments in the skeleton (3). Metabolic turnover of the trabecular bone is very high when compared to the cortical bone. While trabecular bone compartment ensures resistance to compressive stress, cortical bone, characterised by higher bone mineral density, is mainly responsible for withstanding bending forces (1).

4 624 Table 2 The values of Pearson s correlation coefficient among all the investigated parameters of turkey tibia 624 Parameter Bone weight Bone length Td* Cd MvBMD Bvol ACb A Ix MRWT CI Wy Wf Bone weight x Bone length x Td* x Cd x MvBMD x B vol x A Cb x A x Ix x MRWT x CI x Wy x Wf x *Pearson s correlation coefficient between trabecular bone mineral density and all the investigated parameters of the tibia was estimated for the values obtained from 12- and 20- week-old turkeys.

5 625 However, MvBMD value representing mineral density for both the trabecular and cortical bone compartments measured within whole bone sample, reached the highest correlation with maximum elastic strength and ultimate strength of the tibia. The results obtained in the current study are in accordance with data presented by Ferretti et al. (5), which have shown positive correlations between cross-sectional area and moment of inertia with stiffness and strength of the femur in rats. In contrast to our results showing negative correlations between MRWT and all other morphometric, densitometric, and mechanical parameters (except for cortical index) of the tibia, the mentioned above authors have shown positive correlation of this parameter with bone stiffness and strength of the femur. However, their study was performed on rats of both sexes at the age between 18 and 44 weeks. Clearing, morphological differences of the skeleton conditioned by bone modelling and remodelling related to the periods of skeletal development and aging may explain this discrepancy. Negative correlations of MRWT and CI with all other investigated parameters of the tibia point to an inverse relationship of both these parameters with other morphometrical, densitometric, and mechanical properties in the growing turkeys. Considering significant positive correlations among parameters such as Td, Cd, MvBMD, B vol, A Cb, A, Ix, obtained using computed tomography technique, with the values of Wy and Wf, computed tomography may be postulated as a useful tool not only for the determination of volumetric bone mineral density but also for predicting bone mechanical properties. Computed tomography technique also makes possible a precise determination of bone geometrical properties and total bone volume the parameters strictly reflecting bone morphological properties. The data obtained from densitometric and morphometric analyses may be completed with three-dimensional reconstructions of the investigated bone. Furthermore, non-invasive imaging of the internal structure of a bone at any projection may be easily performed. All these data indicate that computed tomography technique has great value for not only investigations of morphometrical and densitometric properties of bones isolated ex vivo or in vivo, but it may also be used to predict bone mechanical endurance. Advantages of this technique give the possibility for precise and noninvasive monitoring of skeletal system properties in vivo during different stages of the development of animals, which is important for optimal breeding selection. 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6 Tatara M.R., Śliwa E., Krupski W., Brodzki A., Pasternak K.: Ornithine alpha-ketoglutarate increases mineralization and mechanical properties of tibia in turkeys. Bone 2006, 39, Tatara M.R., Tygesen M.P., Sawa-Wojtanowicz B., Krupski W., Majcher P., Harrison A.P.: Bone development: the effect of short term alpha-ketoglutarate administration on long term mechanical properties of ribs in ram lambs. Small Rumin Res 2007, 67, Tatara M.R.: Current methods for in vivo assessment of the skeletal system in poultry. Medycyna Wet 2006, 62, Van der Klift M., De Laet C.E., Mccloskey E.V., Johnell O., Kanis J.A., Hofman A., Pols H.A.: Risk factors for incident vertebral fractures in men and women: the Rotterdam study. J Bone Miner Res 2004, 19,

INTERRELATIONSHIPS BETWEEN DENSITOMETRIC, MORPHOMETRIC, AND MECHANICAL PROPERTIES OF THE FEMUR IN MALE POLISH MERINO SHEEP

Bull Vet Inst Pulawy 55, 795-799, 2011 INTERRELATIONSHIPS BETWEEN DENSITOMETRIC, MORPHOMETRIC, AND MECHANICAL PROPERTIES OF THE FEMUR IN MALE POLISH MERINO SHEEP MARCIN R. TATARA, WITOLD KRUPSKI 1, BARBARA