Comparative Evaluation of Biomechanical Testing Methods for Murine Lumbar Vertebral Bodies: Identifying Optimal Techniques for Assessing Bone Structure and Stability

doi:10.21203/rs.3.rs-4761081/v1

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Comparative Evaluation of Biomechanical Testing Methods for Murine Lumbar Vertebral Bodies: Identifying Optimal Techniques for Assessing Bone Structure and Stability

https://doi.org/10.21203/rs.3.rs-4761081/v1

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Numerous genetically modified mouse models are available to evaluate gene functions related to bone metabolism regulation. This study aims to evaluate four prevalent biomechanical testing methods for assessing the structure and stability of murine lumbar vertebral bodies, identifying the most reliable, valid, and reproducible technique. The fourth lumbar vertebrae of 20 female C57BL/6 mice (wild type, 12 weeks) were tested, randomized into four testing groups: Method 1 - compression of the complete lumbar vertebral body (LVB) including the dorsal spinal processi; Method 2 - compression at the vertebra body surface; Method 3 - isolated resection of the vertebral arch; Method 4 - resection of the vertebral arch and straightening of the intervertebral joint surface. Compression was applied using a mono-axial static testing machine. Maximum load, stiffness, yield load, and the elasticity modulus were evaluated. Load-to-failure and yield-to-failure were significantly higher in Method 1. Method 3 showed increased stiffness and significantly increased Young's modulus. The least variation in relative load-to-failure and yield-to-failure was observed with Method 1. Method 4 exhibited the greatest overall variation in specimen values. Method 3 yielded divergent results and is not recommended. Method 1 led to the most consistent and reproducible data and is recommended.

Biological sciences/Biological techniques

Physical sciences/Engineering/Biomedical engineering

Physical sciences/Materials science/Biomaterials/Biomineralization

Physical sciences/Materials science/Biomaterials/Tissues

Animal experiments using mice have the advantage of easy handling, a short reproduction cycle and cheap housing costs due to their size. During the past 20 years, the development of methods to generate genetic modification in mice yielded in a high variety of mouse models to study physiology and pathologies. Thusly, small animal models are indispensable to study the complex mechanisms of bone metabolism and healing. In addition, transgenic mice are a valuable tool to analyze the function of genes in the outcome of diseases [1, 2]. It is also possible to induce osteoporosis in mice using different models.

To determine functional differences introduced by these models in bone, biomechanical testing is an important method in experimental studies to evaluate stability, stiffness and weight bearing capacity [2–4]. In a murine model, size poses to be a significant problem, when analyzing the bone strength in biomechanical studies.

Bone tissue is composed of cortical and cancellous/trabecular areas. Regarding their composition of cortical and trabecular bone tissue and its ratio, bones of the skeleton vary. The biomechanical properties of cortical and trabecular bone are also very different. Analyzing the influence of genes and proteins on bone quality, those two different types of bone structure may be influenced in a very diverse way. Several studies were able to show a distinct influence of certain proteins on cortical bone structure and stability [3–8]. Therefore, different parts of the bony skeleton have to be investigated to distinguish between the effects of targets on cortical and cancellous bone. The vertebral body is characterized by a cancellous bone vs. cortical bone ratio in favor of the cancellous bone. Besides the femoral neck, the lumbar spine is used to measure bone density during diagnosis of bone loss, since changes in bone morphology are early apparent in trabecular bone. Therefore, it is the most established bone to evaluate trabecular bone structure and its changes [7, 9, 10].

The murine lumbar spine consists of six vertebral bodies. The basic morphology shares some similarities to the human lumbar vertebral bodies: ventral- corpus vertebrae, dorsal- arcus vertebrae, surrounding the foramen vertebrale and lateral- two processus transversus. The vertebral joints consist of the processus articularis cranialis et caudalis. The caudal part of the processus articularis overtops the caudal base of the corpus vertebrae [11] (Fig. 1a).

In addition, there are distinct anatomic differences between mice and human vertebral bones. Compared to the human vertebral body (Fig. 1b), the ratio of the murine corpus vertebrae to the arcus vertebrae is smaller; its morphology is long and small due to the lack of axial compression. Compared to the vertebral body the vertebral arch containing the spinal canal is much bigger in mice [12].

Since murine vertebral bodies are just some millimeters in size, the biomechanical testing method must be chosen wisely to obtain valid information. A variety of very different biomechanical testing methods for vertebral bodies are published [1, 2, 4, 8, 9, 13, 14]. They differ in manipulation of the bone before measurement due to the bone morphology and embedding into the testing machine. However, the results are difficult to compare. There is no current standardized testing model for vertebral bodies in literature to date. Although, it becomes even more important due to the numerous murine studies to be able to compare results of different strains and models to each other.

Biomechanical testing determines the reaction of biological material under load. The tissue properties (stiffness and maximum load) describe the extrinsic property of the individual vertebral body, they are determined both by the properties of the tissue and by the size of the sample. The material property (toughness and Young´s modulus) is consistent with the stability of the material “vertebral body”. The tissue properties are normalized by cross-sectional area and are converted to a stress-strain curve from which the properties of the tissue (intrinsic properties) itself can be determined. The ultimate stress is a measure of the material strength. The slope of the stress-strain curve defines the elastic (Young´s) modulus of the tissue, whereas the area under the stress-strain curve is the modulus of toughness, which is the energy required to cause failure of the bone matrix itself, independent of the sample size.

The aim of this study was to evaluate the validity, reliability and reproducibility of different established biomechanical testing methods for murine lumbar vertebral bodies and to identify the most appropriate testing method to serve as a standard. We analyzed the performance, precision and reproducibility of the individual methods on the basis of biomechanical parameters.

1.1 Specimen Preparation

All animal experimental methods are reported in accordance to the ARRIVE guidelines (https://arriveguidelines.org). Landesamt für Natur, Umwelt und Verbraucherschutz, Nordrhine-Westphalia, Germany G67/2006 has approved the study. All mice used in this study were female C57BL/6 littermates genotyped as wild type (wt), kept in family groups in an air-conditioned room at a constant temperature of 20–23°C, and fed a standard laboratory diet with ad libitum access to tap water. The animals were sacrificed at 12 weeks by cervical dislocation, with procedures approved by the local government animal rights protection authorities in accordance with NIH guidelines (Landesamt für Natur, Umwelt und Verbraucherschutz, Nordrhine-Westphalia, Germany G67/2006). The fourth lumbar vertebra (L4) was used, dissected, and thoroughly cleaned of soft tissue and the vertebral disk.

The vertebral bodies were prepared in four different manners before biomechanical compression testing (Fig. 2):

Method 1: The whole vertebral body was placed upright and fixated without bone manipulation using bone cement (Palacos R, Heraeus Kulzer GmbH, Germany) to provide a planar surface and unidirectional force application. The dorsal process was pressed into the cement until the caudal surface was horizontal.
Method 2: The placement and fixation were the same as Method 1, but a smaller diameter compression tool was used, excluding the transversal process.
Method 3: The transversal and dorsal processes were resected, and the remaining vertebral body was fixated into the cement.
Method 4: Similar to Method 3, but the cranial and caudal surfaces were ground to create parallel surfaces, eliminating the need for resin embedding.

1.2 Biomechanical Testing

Prior to biomechanical analysis, sample dimensions were determined. The vertebral arc surface area was approximated to a ring-like surface, and the caudal end plate to an ovoid area for calculating Young’s modulus. The change in length as a function of force was used to determine load to yield and load to failure. Values were normalized by setting the mean to 100%.

Biomechanical testing was conducted with a compression testing machine (Lloyd LRK5, Lloyd Instruments), measuring applied force at a rate of 8 kHz using a 250 N load cell with 0.5% accuracy at forces over 5 N, with a velocity of 1 mm/min. The force-displacement curve was visualized using Nexigen software (AMETEK Lloyd Instruments). Load to failure was defined as the maximum load point, and yield load as the turning point between elastic and plastic deformation (Fig. 3a). Stiffness was calculated from the linear part of the curve, and energy and elasticity moduli were derived from the data. Data were compared to recently described methods for evaluating the stability of murine lumbar vertebral bodies.

1.3 Statistical Analysis

One-way ANOVA followed by Tukey's multiple comparisons test was performed using GraphPad Prism version 10.0.0 for Windows (GraphPad Software, Boston, MA, USA). P-values were classified as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Standard deviation and statistical error were also calculated to assess data variation.

1.4 Data Availability

Data is provided within the manuscript or supplementary information files.

2.1 Determination of Absolute Biomechanical Properties

Maximum Load

The numerical values decreased with increasing levels of manipulation (Fig. 3b) Without any manipulation of the bone (method 1), the maximum load of the lumbar vertebrae in mice was 38.3 N. With methods 2 and 3, the values decreased to 20.8 N and 20.3 N, respectively, which is approximately 70% of the result from method 1. Using the maximal manipulation method (method 4), the value further decreased to 15.5 N, representing 50% of the maximum value measured with method 1.

Yield Load

Determination of the yield load revealed similar trends. The highest values were measured using method 1 (25.9 N), compared to method 2 (18.2 N), which is 70% of the result from method 1. The values further decreased with methods 3 and 4, yielding 12 N. There is a significant difference between method 1 and the other testing methods in both maximum and yield load (Fig. 3c).

Stiffness: The stiffness of the vertebrae using method 1 was 95 N/mm. Methods 2 and 4 produced very similar stiffness values (method 2: 103.9 N/mm; method 4: 93.4 N/mm). The mean stiffness value obtained using method 3 was 127 N/mm, higher than any other method tested. Although there was no significant difference between all tested methods, it was notable that the standard deviations for methods 3 and 4 were higher compared to methods 1 and 2 (Fig. 3d).

Young’s Modulus: The material constant of the vertebra obtained by method 3 (232 N/mm) was significantly higher compared to the methods where the vertebral body remained intact (method 1: 101 N/mm; method 2: 130 N/mm). This increase was also evident in the stiffness but not in the load to failure, suggesting that the displacement in method 3 might be altered compared to other methods. Interestingly, method 4 (145 N/mm) did not show much alteration compared to the Young’s modulus obtained by methods 1 and 2 (Fig. 3e).

4.2 Determination of Relative Biomechanical Properties

To determine the relative deviation and reproducibility of each method, we calculated the experimental values in proportion to the mean, setting the mean value to 100%. We marked thresholds at 25% and 50% divergence from the mean to visualize aberrations (Fig. 4). In the box-whisker plot, the box represents the lower and upper quartiles of the values, encompassing the range where 4 of the 5 replicates are located. This conversion visualizes the reproducibility of the methods for given parameters.

For single-point parameters (load to failure and load to yield; Fig. 4a and b), method 1 showed the smallest deviation. Particularly for load to yield, methods that kept the bone intact demonstrated good reproducibility compared to methods 3 and 4. We investigated the slope of the force/displacement curve to present the stiffness and calculate the Young’s modulus (Fig. 4c and d). The overall reproducibility was lower for these parameters. It should be noted that for these calculations, experimental values such as displacement and force for stiffness, and force, displacement, and diameter for Young’s modulus were required, each with individual measurement errors. Despite these factors, method 1 exhibited the lowest deviation across all the biomechanical parameters obtained.

The aim of this study was to identify a valid and reproducible method to evaluate the biomechanical properties of murine lumbar vertebral bodies. Our findings demonstrated that the vertebral body, characterized by a high cancellous-to-cortical bone ratio and active bone remodeling [7], is a suitable model for early detection of changes in bone quantity and quality. To determine the biomechanical properties of trabecular bone, compressive force is the most physiological testing method [15], while bending and torsion forces are more suitable for testing cortical bones [6, 16]. However, the lack of standardization in applying compressive force to vertebrae and measuring forces presents challenges.

In this study, we compared four biomechanical testing methods to evaluate their validity and reproducibility. Our results indicated that Method 1, which involves minimal manipulation of the vertebral body, provided the most consistent and reproducible data. This method maintains the integrity of the vertebral body, ensuring that the force is applied uniformly across the entire structure.

Interestingly, our findings align with Goetzen et al. (2005), who also tested vertebral bodies using an unmodified bone approach similar to Method 1. Their results, derived from 12-month-old C57BL/6 mice, showed consistency with our findings, albeit with a different age group. This consistency across different ages highlights the robustness of Method 1.

Tommasini et al. (2005) used a modified Method 2 with 15-month-old mice, involving grinding the cranial and caudal surfaces and fixing the vertebral body with a rod through the vertebral canal. They reported a higher median maximum load (28.5 N) compared to our results (20.75 N) with 12-week-old mice. This discrepancy could be attributed to the age difference, as bone strength increases with age. In our previous studies with 16-month-old mice, we observed a similar median maximum load of 27 N.

Almeida et al. (2007) used Method 3, removing the vertebral arch and adjusting the inclined surfaces with a custom-made flexible bearing area. Their studies with 8- and 16-week-old mice did not provide directly comparable data due to age differences. However, their approach highlights the variability introduced by extensive sample manipulation.

Akhter et al. (2001) employed a method similar to our Method 4 on 4-month-old C57BL/6 mice, finding results consistent with our study regarding maximum load, yield load, and stiffness. Their findings support the notion that extensive manipulation can still yield valid results, though our study suggests Method 1 is more reliable overall.

Comparing all four methods, we observed that Methods 2–4, which involved partial vertebral body usage, resulted in lower absolute load to failure and yield load values. However, normalized parameters like stiffness and Young’s modulus from Methods 2 and 4 were comparable to those of Method 1. Method 3 was an outlier, particularly in stiffness and Young’s modulus, likely due to displacement issues during testing.

By normalizing measured values to the mean, Method 1 exhibited the lowest deviation, while Method 4 showed the highest. Method 4's extensive manipulation increased error risk during sample preparation. Although Method 3 produced consistent results among samples, it likely contained a systematic error.

This study has limitations, including the young age of the mice (12 weeks) and a small sample size (five per method). The age-related decline in trabeculae, common in older mice, was not considered, which could be relevant for Methods 3 and 4 that involve cortical damage during preparation. Despite these limitations, our results consistently support the validity of Method 1, especially for stiffness and Young’s modulus.

Overall, we recommend Methods 1, 2, and 4 for obtaining comparable data on the compression strength of murine lumbar vertebral bodies. Method 1, requiring the least sample manipulation, provided the most consistent data. Interestingly, Method 1 is the least frequently used in literature, with Methods 2 and 4 being more common. This underscores the necessity for comparative studies like ours to standardize biomechanical testing procedures, enhancing reproducibility and reducing animal numbers.

In this study, we compared four different methods of preparing murine lumbar vertebrae to test their resistance towards compression in a material testing machine. With just embedding the whole caudal surface for even stand and applying force over the vertebral body and the transversal processes (method 1), we identified the most reliable and reproducible biomechanical testing procedure for lumbar vertebrae and could clearly demonstrate that the extent of manipulation to isolate parts of the bone or fix it has an impact on the outcome of the results. The more the bone was modified, the standard deviation of the values increased pointing toward decrease of validity and reproducibility.

Our results emphasize the need of standardized biomechanical testing procedures for bone in animal experiments in order to maximize significance and reproducibility and minimize animal numbers.

Author Contribution

Author contribution: Conceptualization DK, BW, RS; Data curation DK and MT Formal analysis DK, BW, SS and HH; Funding acquisition MR and RS; Investigation BW, SS and HH; Methodology BW, SS and HH; Project administration MT and RS; Resources MR and RS; Supervision RS; Validation BW, MT, RS; Visualization; DK and BW Roles/Writing - original draft DK and BW; and Writing - review & editing DK, MT and RS

Acknowledgement

The authors thank Simone Niehues for her technical support.

Data Availability

Data is provided within the manuscript or supplementary information files.

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No competing interests reported.

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You are reading this latest preprint version

Comparative Evaluation of Biomechanical Testing Methods for Murine Lumbar Vertebral Bodies: Identifying Optimal Techniques for Assessing Bone Structure and Stability

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

1.1 Specimen Preparation

1.2 Biomechanical Testing

1.3 Statistical Analysis

1.4 Data Availability

Results

2.1 Determination of Absolute Biomechanical Properties

4.2 Determination of Relative Biomechanical Properties

Discussion

Conclusion

Declarations

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1