Improving results in rat fracture models: enhancing the efficacy of biomechanical testing by a modification of the experimental setup

Abstract

BackgroundAnimal fracture models, primarily performed in rats, are crucial to investigate normal and pathological bone healing. However, results of biomechanical testing representing a major outcome measure show high standard deviations often precluding statistical significance. Therefore, the aim of our study was a systematical examination of biomechanical characteristics of rat femurs during three-point bending. Furthermore, we tried to reduce variation of results by individually adapting the span of bearing and loading areas to the bone’s length.MethodsWe examined 40 paired femurs of male Wistar-rats by DXA (BMD and BMC of the whole femur) and pQCT-scans at the levels of bearing and loading areas of the subsequent biomechanical three-point bending test. Individual adjustment of bearing and loading bars was done respecting the length of each specimen. Subgroups of light (< 400 g, n = 22) and heavy (> 400 g, n = 18) animals were formed and analysed separately. We furthermore compared the results of the individualised bending-setting to 20 femurs tested with a fix span of 15 mm.ResultsFemurs showed a length range of 34 to 46 mm. The failure loads ranged from 116 to 251 N (mean 175.4 ± 45.2 N; heavy animals mean 221 ± 18.9 N; light animals mean 138.1 ± 16.4 N) and stiffness ranged from 185 N/mm to 426 N/mm (mean 315.6 ± 63 N/mm; heavy animals mean 358.1 ± 34.64 N/mm; light animals mean 280.8 ± 59.85 N/mm). The correlation of densitometric techniques and failure loads was high (DXA R2 = 0.89 and pQCT R2 = 0.88). In comparison to femurs tested with a fix span, individual adaptation of biomechanical testing homogenized our data significantly. Most notably, the standard deviation of failure loads (221 ± 18.95 N individualized setting vs. 205.5 ± 30.36 N fixed) and stiffness (358.1 ± 34.64 N/mm individualized setting vs. 498.5 ± 104.8 N/mm fixed) was reduced by at least one third.ConclusionsTotal variation observed in any trait reflects biological and methodological variation. Precision of the method hence affects the statistical power of the study. By simply adapting the setting of the biomechanical testing, interindividual variation could be reduced, which improves the precision of the method significantly.Electronic supplementary materialThe online version of this article (10.1186/s12891-018-2155-y) contains supplementary material, which is available to authorized users.

Investigation of the effect of ghrelin on bone fracture healing in rats

“…The results of the biomechanical test showed that at the end of the recovery period of 28 days the groups treated with ghrelin had a statistically higher breaking force than the control groups. The breaking force results of the ghrelin‐treated group was as high as breaking force needed for a healthy rat femur that was reported as 116 to 251 N in the study of Prodinger et al 17 Ghrelin treatment through 28 days was shown to improve fracture healing in rat femur compared to the no‐treatment group. Compared to healthy bone, breaking distances of the fracture model groups were higher.…”

Influence of bone defect position and span in 3-point bending tests: experimental and finite element analysis

“…Each experimental model represent different mechanisms, where bone repair of cortical perforation defects occurs by intramembranous ossification, and bone repair of fracture is the result of abundant cartilaginous callus formation followed by endochondral ossification. 3 Various experimental studies focused on the diaphysis of long bones, 1,2,4 however to evaluate biomechanical effect of bone alterations, the metaphysis of a femur or tibia should be considered, since the diaphysis of long bones consist only of cortical bone and bone marrow and major changes due to bone degenerative diseases are also observed in the trabecular bone that is located in the metaphysis. 3,5,6,7,8,9 In order to determine the effect of any therapy during the repair process of bone defects, it is essential to evaluate the structural fracture resistance.…”

“…3 Various experimental studies focused on the diaphysis of long bones, 1,2,4 however to evaluate biomechanical effect of bone alterations, the metaphysis of a femur or tibia should be considered, since the diaphysis of long bones consist only of cortical bone and bone marrow and major changes due to bone degenerative diseases are also observed in the trabecular bone that is located in the metaphysis. 3,5,6,7,8,9 In order to determine the effect of any therapy during the repair process of bone defects, it is essential to evaluate the structural fracture resistance. Several structural parameters such as geometry, mineral content and density are used to indirectly evaluate the biomechanical properties of bone, nonetheless, bone ability to resist fracture can only be assessed by biomechanical strength tests.…”

“…11 Three-point bending tests are the most commonly used mechanical tests to characterize bone material and biomechanical properties of long bones. 12,13 Standardization of three-point bending tests on the methaphysis of long bones without bone defects has been reported, 3,7,14 however, there there are no guidelines for parameters such as span and bone defect positioning. 12,13,14 Adequate stability of the bone between the supports during the test must is determined by the length of metaphysis region, which constitutes a limitation for the span distance.…”

“…A standardized protocol is needed to allow comparisons of data from different sources and to ensure that biomechanical properties are obtained at the targed area of the bone defect. 3,7,14 Three-point bending tests provide structural information about the whole bone, such as stiffness and strength. However, they cannot provide local information, such as stress distribution.…”