Bone tissue research is becoming a progressively more interesting field of research for multiple medical specialties such as orthopedic-, trauma- and plastic surgery, but also for osteology and endocrinology due to the various changes in bone homeostasis caused by systemic diseases. An elemental part of all these research studies is the assessment of the quantitative properties of bone, which are extensively evaluated by biomechanical testing technologies.
Long bones consist of three main parts: the diaphysis, consisting mainly of cortical bone, the metaphysis, containing mainly cancellous bone, and the epiphysis. Cancellous bone is extremely sensible to changes in bone-mineral homeostasis and is therefore the area first affected by developing osteoporosis. To biomechanically evaluate these areas, special aspects and requirements have to be considered. First, a stable fixation of the bone has to be assured to avoid sudden changes in position while the stamp drives down during force application. To address this aspect, various possibilities have been designed. In previous studies, one approach was to fix the rat bone, a rat femur in these cases, but we consider it nonetheless just as relevant for the rat tibia, on a metal block with a proximal deepening in which the proximal bone could be placed during testing [6, 7, 29]. The shaft was located between to cylinders capable of rotation [6, 7, 29]. The distal end was placed onto the plane surface of the block [6, 7, 29].
Secondly, the shape and dimensions of the stamp are important. It should not apply the force only to a point shaped area, but in a transversal manner across the metaphyseal area in order to apply the force in an evenly distributed fashion. We therefore used the above mentioned design (Figure 4). Thirdly, it has to be made sure that during testing only the metaphyseal part is analyzed, and that the results are not influenced by the diaphyseal part of the bone. This influence of the diaphyseal part is the critical aspect which could, until now, not be eliminated via the existing testing devices. The distal part of the tibia moves over a fixed metal block during diaphyseal lengthening [2, 18, 30, 31]. By moving in this described manner, we propose that the distal diaphysis was transferring the arising frictional forces created by this movement of bone over the aluminum block to the stiffness and yield load properties of the metaphyseal area.
In order to eliminate this falsification caused by the arising frictional forces during testing, there is a need to position the distal diaphysis on a surface that allows a resistance-free gliding during testing.
To achieve this, we decided on a mobile, ball-mounted technique.
During biomechanical testing, a lengthening of the rat diaphysis occurs of about 0.2-0.3 cm. In order to ensure a stable gliding of the mobile, ball-mounted platform, measuring 3.8 cm × 4 cm × 0.9 cm (length × width × height) with a top area of 1.4 cm × 4 cm (length × width), we used a consecutive series of 5 stainless steel balls on each side with a distance of 0.5 cm between the balls. To fully eliminate friction, a special silicone based ball bearing fat called “Plastilube” (Henkel AG, Duesseldorf, Germany) had to be applied to the balls.
For our study, we regarded the recently used techniques [2, 18, 30, 31] as state of the art, and respectively as a negative control. Thus, the testing of Group1 was performed according to their described methodologies, in which the distal tibia diaphysis is positioned on a fixed aluminum block [2, 18, 30, 31].
The differences of our results between the two plate designs are apparent. The stiffness of group 1 was around 20 percent higher than the stiffness of Group 2. Since the term stiffness describes the resistance of a tissue against an incoming force [28], these results demonstrate the influence of the frictional forces arising from the movement of the distal diaphysis over a fixed metal plate on the metaphyseal biomechanical properties. Thus, the measured results for the stiffness are falsified by these frictional forces. In terms of the yield load, this falsification is even more evident. Here the change from elastic to plastic deformation under the applied force is significantly enhanced in Group 1. Interestingly, the results found in the final part of the testing procedure, the failure load, were very similar. Thought should also be given to the applicability of these findings in regard to larger animal models as well as further examinations of cancellous bone of the metaphysis of, for example, the humerus, the femoral neck or the distal radius.
Further we do not want to hesitate to mention the limitations of this study. One limitation would be that, even though using bones from the same animal in comparison is a generally accepted method (since the conditions such as nourishment, exercise, mechanical strain, environmental conditions etc. are as much the same as possible for the animal and thus for the bones), the procedure of comparing the bones of the same animal to one another do not have an independent confirmation. Therefore it cannot be proven that the tibiae of the same rat have a comparable bone architecture and strength. For similarity of the bones to be not just highly possible but proven, there should be, in future experiments, an investigation via micro CT, BMD, or a measurement of trabecular bone quantity.
Other limitations of this study could be improved with a micro-camera. We did not utilize one because this was a project purposed to identify if there was at all an influence and thus a difference in the two setups concerning friction during the three-point bending/breaking test. Now, considering the presented results, we recommend the utilization of a micro-camera in future follow-up experiments to go into more detail on the influence of friction on the outcome of the three-point bending/breaking test. For example, had we had data from a micro-camera, it would have been possible to record the exact distance of the diaphyseal lengthening on the aluminum block and so to produce force-displacement curves of this matter. Also, we could have numerically assessed in which phase of loading most of the diaphyseal lengthening occurred. Macroscopically most of it occurred in the early phase of loading, which we attribute to the elasticity the bone displays, before it, under further loading, transits into the phase of plastic deformation. Another limitation concerning the lack of a micro-camera would be the fact that with one, it might be possible to calculate such values as the frictional coefficient, the frictional force itself, and the contribution of friction to the experiment. Therefore, again, we suggest this to be included in follow up studies.
It would be interesting, since the animals used in this study did not suffer from the condition of osteoporosis and since osteoporosis is the most common disease which requires testing of the metaphyseal area of long bones, to examine osteoporotic bone with both presented testing devices. Unfortunately this suggested approach was not covered by our animal protection committee in the case of this study. However, we would like to potentially follow up on this matter.
