An experimental test method for estimating the bending strength and disturbance of rat femur

Abstract

The invention discloses an experimental test method for estimating the bending strength and disturbance of rat femur. The bending strength of rat femur is obtained by a high-speed camera and a three-point bending test. Subsequently, an electron microscope scanning image can be binarized to obtain the precise cross-sectional area of ​​the rat femur fracture surface. The bending strength and disturbance of the rat femur are calculated according to a material mechanics formula. Then, the biomechanical performance of the rat femur is evaluated. The method can well characterize the influence of modeling treatments with different blood sugar concentrations on the vital signs of the rat from a mechanical point of view. The method can accurately calculate experimental data such as the bending strength, disturbance, displacement field and strain field of the rat femur fracture surface. The tested rat femur mechanical strength data is wide and the test accuracy is high. The biomechanical performance of the rat femur can be evaluated in many aspects.

Description

Technical Field

[0001] The invention relates to the technical field of rat femur biomechanical strength testing, in particular to an experimental testing method for estimating the bending strength and disturbance of rat femur. Background Art

[0002] There are many manifestations of the biomechanical strength characteristics of rat femur, such as tensile strength, compressive strength, torsional strength and bending strength. Some scholars have conducted various explorations on the test methods of tensile strength, compressive strength and torsional strength of rat femur. However, these mechanical strengths are somewhat different from the stress characteristics in real life. For example, the Chinese invention patent with application number 201610102091.9 discloses a stress loading device, loading system and method for constructing a stress fracture animal model. In this application, the moving wheelbase of the tissue fixation unit is adjusted by a linear guide rail so that the bone to be tested of the experimental animal is placed in the tissue fixation unit. The displacement and speed of the linear actuator movement are set by the manual control part of the LabVIEW control program to apply a certain preload.

[0003] The characterization of the bending strength of rat femur is the most common aspect, and there are still a lot of gaps in the accurate measurement of the bending strength of rat femur. On the one hand, because there is part of the bone marrow inside the rat femur, it is generally a hollow structure, and the influence of the hollow section of the femur on the bending strength needs to be fully considered. This requires accurate measurement of the cross-sectional area of ​​the femur, and then characterization of the inertial cross-sectional area of ​​the femoral fracture surface. On the other hand, when the human femur is subjected to a certain impact force or other load, the force characteristics of the femur are in a three-point bending state or an eccentric bending state. For this reason, the force state of the rat femur can also be idealized to be similar to that of the human femur. Therefore, the three-point bending experiment is generally used for the characterization of the bending strength of the rat femur, especially the bending strength test experiment. The simultaneous spatial evolution test of the displacement field and strain field of the mouse femur with a high-speed camera is rarely carried out, and the accurate test of the bending strength and disturbance of the rat femur cannot be achieved.

[0004] Therefore, the present invention provides a new solution to solve this problem. Summary of the invention

[0005] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide an experimental test method for estimating the bending strength and disturbance of rat femur.

[0006] The technical solution is: an experimental test method for estimating the bending strength and disturbance of rat femur, the method comprising the following steps:

[0007] 1) preparing a number of normal rats, and performing type 2 diabetes modeling treatment on some of the rats to obtain a model group, and then dividing the modeled rats into three groups on average, and feeding two of the groups with Chinese medicine reagents and Western medicine reagents respectively, and then obtaining four groups of rat models with different blood glucose concentrations, namely, a normal group, a model group, a Chinese medicine group, and a Western medicine group;

[0008] 2) The femurs of the four groups of rat models in step 1) were collected, the muscle tissues were removed, and then the femurs were placed in a phosphate buffered saline solution for culture treatment;

[0009] 3) The rat femurs were taken out from the phosphate buffered saline solution and dried, and then black and white speckles were sprayed on the upper surface of the femurs of the four groups of rats for digital image correlation processing;

[0010] 4) The femurs of the four groups of rats were placed on a compression load loading device for a three-point bending test. A high-speed camera was used to take real-time photos of the fracture process of the femurs of the rats, and the test data were imported into a computer for processing;

[0011] 5) deriving a rat femur load time history curve according to the test data obtained in step 4), and analyzing the displacement field and strain field between the fracture points of the rat femur;

[0012] 6) Scan and analyze the rat femoral fracture surface using an electron microscope, input the scanned image information into Image J software for binarization processing, and after the conversion is completed, the diameter and area of ​​the rat femoral fracture surface can be accurately calculated based on the black and white pixel points of the binary image and the scale ratio of the electron microscope scanned image, and then the bending strength is calculated according to the bending strength theoretical formula of material mechanics, and then the bending strength and disturbance analysis are performed in combination with the image information of the high-speed camera;

[0013] 7) Based on the analysis of the bending strength of the femurs of the four groups of rats, the biomechanical properties of the rat femurs were evaluated by mechanical strength.

[0014] Furthermore, in step 1), the Chinese medicine reagent and the Western medicine reagent are respectively a ginseng and astragalus compound reagent and a saxagliptin tablet reagent.

[0015] Furthermore, the specific analysis process in step 5) is as follows:

[0016] a. Randomly select the test point P(x i ,y i ), and the test point P(x i ,y i ) is used as the center to select a square sub-image with a size of (2N+1)×(2N+1) pixels, namely, the reference subset;

[0017] b. Expressed as f(x,y); at the same time, from the deformed image, a square sub-image (M>N) with a size of (2M+1)×(2M+1) pixels is selected with the corresponding pixel point as the center, that is, the search subset;

[0018] c. Randomly select a deformed subset in the search subset, denoted by g′(x′,y′), and the size of the deformed subset is (2N+1)×(2N+1) pixels;

[0019] d. Then calculate the correlation between f(x, y) and g′(x′, y′) according to the correlation coefficient formula to obtain the correlation coefficient distribution diagram of the whole field;

[0020] e. The peak of the correlation coefficient distribution graph is the best matching point between f(x, y) and g′(x′, y′); and the center point P′(x i ′,y i The difference between the coordinates of the center point of the reference subset and the coordinates of the test point P(x i ,y i )’s displacement vector;

[0021] f. Using the same method, the above related operations are performed on each pixel point in the entire speckle pattern to obtain the full-field displacement and full-field strain of the rat femur, and then the displacement field and strain field of the rat femur can be further analyzed.

[0022] Furthermore, the compression load applying device in step 4) is an electro-hydraulic servo press equipped with a three-point bending fixture.

[0023] Furthermore, the three-point bending test in step 4) can be a central symmetrical loading or an eccentric asymmetrical three-point bending loading.

[0024] Through the above technical solution, the beneficial effects of the present invention are:

[0025] 1. The range of test data is wide; the present invention can accurately calculate the experimental data such as the bending strength, disturbance, displacement field and strain field of the rat femoral fracture surface, and the tested rat femoral mechanical strength data is wide. The disturbance of the rat femur can be tested by high-speed camera combined with digital image correlation method, and compared and analyzed with the theoretical value of the theoretical formula, and finally the bending strength of the rat femur can be corrected; in addition, this test range can not only be extended to the measurement of rat femur, but also can be applied to the test of bones of biological materials such as rabbits;

[0026] 2. High test accuracy: The present invention can accurately test the displacement field and strain field data of the rat femur fracture point, which has always been the limitation of rat bone experimental test data. The displacement field and strain field accuracy of the rat femur can reach 0.001mm.

[0027] 3. Evaluate the biomechanical properties of rat femur from multiple aspects; the testing method of the present invention can not only evaluate the biomechanical properties of rat femur from the bending strength of rat femur, but also evaluate the biomechanical properties of rat femur according to the disturbance value of rat femur, and can perform mutual verification and evaluation from two aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a flow chart of the testing method of the present invention;

[0029] Figure 2 It is a schematic structural diagram of the compression load loading device of the present invention;

[0030] Figure 3 Schematic diagram of the force-displacement curve of the femur of rats in the normal group;

[0031] Figure 4a Schematic diagram of loading points of femur of rats in normal group;

[0032] Figure 4b This is a schematic diagram of the calculation of the bending moment at the loading point of the femur of the normal group rats;

[0033] Figure 4c It is a schematic diagram of the cross section of the femur of the normal group rats;

[0034] Figure 5a This is the electron microscope scanning image of the femur of the normal group rats;

[0035] Figure 5b This is the electron microscope scanning binary image of the femur of the normal group rats;

[0036] Figure 6 Schematic diagram of the displacement time course curve of the monitoring points on the loaded femoral section of the femur of the normal group rats. DETAILED DESCRIPTION

[0037] The above and other technical contents, features and effects of the present invention are described in detail below with reference to the attached Figure 1 To Attachment Figure 6 It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0038] The application principle of the present invention is described in detail below in conjunction with the following embodiments.

[0039] Embodiment 1:

[0040] 1) Three healthy male Wistar normal rats aged 10-13 weeks and nine healthy male GK rats aged 10-13 weeks were selected and fed adaptively for two weeks. During this period, random blood glucose was measured once to understand the blood glucose status of the rats. The feeding environment temperature was 19-22°C, the relative humidity was 50%-70%, and the 12-hour light / dark circadian rhythm was maintained (darkness, 7:00am to 7:00pm; light, 7:00pm to 7:00am in the afternoon). All rats were allowed to drink water and eat freely.

[0041] Establishment of type 2 diabetes model group: Wistar rats were fed with ordinary growth and breeding feed as normal control group, and the formula of ordinary growth and breeding feed was: 66% carbohydrate, 22% protein, 12% fat, and all GK rats were fed with high-fat feed, and the formula of high-fat feed was: 88.2% ordinary animal feed, 10% refined lard, 1.5% cholesterol, 0.3% pig bile salt; random blood glucose of each group of animals was measured 1-2 times a week (the frequency was adjusted according to the blood glucose value), until the blood glucose value of the model GK rats was ≥11.1mmol / L for 3 consecutive times, thus the type 2 diabetes model group was established;

[0042] The 9 GK rats included in this experiment were randomly divided into model group (n=3), western medicine group-saxagliptin group (n=3), and traditional Chinese medicine group-ginseng and astragalus compound group (n=3) according to blood sugar;

[0043] 2) After killing the animals, the femurs of the rats in the model group (type 2 diabetes), western medicine group (saxagliptin tablets), Chinese medicine group (Shenqi compound) and normal group were separated, the muscle tissue was removed, and the specimens were wrapped with gauze wetted with physiological saline. The femurs of the rats after sampling were placed in a phosphate buffered saline solution with a temperature of 4°C and a pH value of 7.2-7.4 for preservation;

[0044] 3) Before the three-point bending test, the femurs of the rats in the model group (type 2 diabetes), western medicine group (saxagliptin tablets), Chinese medicine group (Shenqi compound) and normal group were taken out from the phosphate buffered saline solution for drying, and then sprayed with black and white scattered spots for processing by digital image correlation method;

[0045] 4) The femur of the normal group rats was placed on the three-point bending fixture of the electro-hydraulic servo press, with the upper end contacting the loading head 2 and the lower end contacting the two support brackets 3. The distance between the two ends of the loading support was set to l = 25 mm according to the rat femur. Figure 2 As shown;

[0046] 5) The hydraulic loading system 4 is used to load the load, and the loading rate is selected to be 0.1 mm / min. The computer 7 is used to automatically collect data on the force and displacement, and the collection frequency is 60 Hz. Then, the load-stress time-history curve of the rat femur can be obtained. Further, the peak load value of the rat femur fracture can be obtained according to the load-time-history curve of the rat femur, and the load peak value is 126.61 N. Figure 3 As shown;

[0047] 6) The rat femur is idealized and simplified to a three-point bending simply supported beam stress state, and its bending moment envelope diagram is obtained, as shown in Figure 4a and Figure 4b As shown, C is the loading point, x is the distance between the loading head and the left support A of the simply supported beam, a is the distance between the loading point and the left support A, b is the distance between the loading point and the right support B, l is the distance between the left and right supports, and F R is the load value applied by the loading head, F RA and F RB is the support reaction force of the left and right supports, and when the loading force of the electro-hydraulic servo press is 1N, the maximum bending moment of the three-point bending loading point C can be expressed as ab / l; if the embodiment is centrally symmetrically loaded, a=b=12.5mm, and the maximum bending moment of the loading point C is M=0.791N·m;

[0048] 7) The fracture surface of the rat femur was scanned by electron microscope and then input into ImageJ software for binarization to obtain the outer diameter D1 and inner diameter D2 of the rat femur, as shown in Figure 4c As shown, the cross-sectional area of ​​the rat femur fracture surface can then be calculated as A = (πD1 2 -πD2 2 ) / 4=4.83mm 2 , and the cross-sectional bending coefficient is shown in formula (1), as Figure 5a and Figure 5b As shown;

[0049]

[0050] In the formula, D1 is the outer diameter, α is the ratio of the inner diameter to the outer diameter;

[0051] Then, according to the material mechanics bending strength formula (2), the maximum bending stress value of the bone fracture surface in the normal group can be obtained to be 0.493MPa, and the formula expression is as follows:

[0052]

[0053] 8) According to formulas (1) and (2), the bending stress at the loading point is calculated to obtain the allowable stress value range [σ] of the rat femur. Then, the disturbance ω of the loading point is calculated according to the load value. The disturbance formula is shown in formula (3). Then, the allowable displacement value u1=ω of the rat femur is obtained. C =0.52mm;

[0054]

[0055] in E represents the elastic modulus of rat femur, F P It represents the load value on the femur of rat;

[0056] 9) Based on the image information obtained by the high-speed camera 5 and the lighting system 6, the displacement value u2=0.50 mm tested during the loading process is obtained by the numerical image correlation (DIC) system 7, such as Figure 6 As shown, by comparing and analyzing the error range of u1 and u2 values ​​to the average value, a more accurate maximum perturbation allowed for rat femur is 0.51 mm;

[0057] Embodiment 2-4:

[0058] Repeat steps 3) to 9), and obtain the maximum bending stress values ​​of the femurs of rats in the model group, the Chinese medicine group, and the Western medicine group according to steps 7) and 9), and the maximum disturbance value of the femurs of rats obtained in the ninth step, as shown in Table 1. The biomechanical properties of the femurs of rats under different blood glucose concentrations were analyzed from the aspects of stress and displacement, and then the biomechanical properties of the femurs of rats were analyzed from a biomechanical perspective.

[0059] Table 1 Experimental data of bending strength and maximum disturbance value of rat femur in different groups

[0060]

[0061]

[0062] The evaluation method of femoral pathological mechanism of rats after different modeling treatments is as follows:

[0063] If the maximum bending stress values ​​of the femurs of rats in the western medicine group (saxagliptin tablets) and the Chinese medicine group (Shenqi compound) are less than that of the normal group and greater than that of the femurs of rats in the model group (type 2 diabetes), it means that the drug treatment has an improving effect on the biomechanical properties of the femurs of rats; if the maximum bending stress values ​​of the femurs of rats in the western medicine group (saxagliptin tablets) and the Chinese medicine group (Shenqi compound) are less than the maximum bending stress values ​​of the femurs of rats in the model group (type 2 diabetes), it means that the drug treatment has an attenuating effect on the biomechanical properties of the femurs of rats; if the maximum bending stress value of the femurs of rats in the western medicine group (saxagliptin tablets) is greater than the maximum bending stress value of the femurs of rats in the Chinese medicine group (Shenqi compound), it means that the western medicine group has a greater effect on improving the biomechanical properties of the femurs of rats than the Chinese medicine group, and vice versa;

[0064] If the maximum perturbation value of the femur of rats in the Chinese medicine group and the Western medicine group is less than that of the normal group and greater than the maximum bending maximum perturbation value of the model group (type 2 diabetes), it means that the drug treatment has an improving effect on the biomechanical properties of the rat femur; if the maximum perturbation value of the femur of rats in the Chinese medicine group and the Western medicine group is less than the maximum bending maximum perturbation value of the femur of rats in the model group (type II diabetes), it means that the drug treatment has an attenuating effect on the biomechanical properties of the rat femur; if the maximum perturbation value of the femur of rats in the Western medicine group (saxagliptin tablets) is greater than the maximum perturbation of the femur of rats in the Chinese medicine group (ginseng and astragalus compound), it means that the Western medicine group has a greater effect on improving the biomechanical properties of the rat femur than the Chinese medicine group, and vice versa;

[0065] The experimental results showed that the maximum bending stress values ​​of the femurs of rats in the Chinese medicine group (0.418MPa) and the Western medicine group (0.408MPa) were less than 0.493MPa and greater than 0.377MPa, indicating that the drug treatment had an improvement effect on the biomechanical properties of the rat femurs, but would not exceed that of the normal group; the bending strength value of the femurs of rats in the Chinese medicine group was 0.418MPa, which was greater than that of the Western medicine group (0.408MPa), indicating that the Chinese medicine group had a better improvement effect on the biomechanical properties of the rat femurs;

[0066] The maximum disturbance value of the femur of rats in the Chinese medicine group (0.432mm) and the western medicine group (0.422mm) was less than 0.510mm and greater than 0.377mm, indicating that the drug treatment had an improvement effect on the biomechanical properties of the rat femur, but would not exceed that of the normal group; the maximum disturbance value of the femur of rats in the Chinese medicine group was 0.432mm, which was greater than 0.422mm in the western medicine group, indicating that the Chinese medicine group had a better improvement effect on the biomechanical properties of the rat femur.

[0067] In summary, the present invention provides an experimental test method for estimating the bending strength and perturbation of rat femur. The bending strength of the rat femur is obtained by a high-speed camera and a three-point bending test. The electron microscope scanning image is then binarized to obtain the precise cross-sectional area of ​​the rat femur fracture surface. The bending strength and perturbation of the rat femur are calculated based on the material mechanics formula, and then the biomechanical properties of the rat femur are evaluated. This can well characterize the effects of different blood glucose concentration modeling treatments on the vital signs of rats from a mechanical perspective.

[0068] The invention can accurately calculate experimental data such as bending strength, disturbance, displacement field and strain field of the fracture surface of rat femur, and the tested mechanical strength data of rat femur is wide. The disturbance of rat femur can be tested by high-speed camera in combination with digital image correlation method, and compared and analyzed with the theoretical value of theoretical formula, and finally the bending strength of rat femur can be corrected; in addition, this test range can not only be extended to the measurement of rat femur, but also can be applied to the test of bones of biological materials such as rabbits; the displacement field and strain field data of rat femur fracture point can be accurately tested, which has always been the limitation of experimental test data of rat bones, and the displacement field and strain field accuracy range of rat femur can reach 0.001mm; the testing method of the invention can not only evaluate the biomechanical properties of rat femur from the bending strength of rat femur, but also evaluate the biomechanical properties of rat femur according to the disturbance value of rat femur, and can perform mutual verification and evaluation from two aspects.

[0069] The above is a further detailed description of the present invention in combination with a specific implementation method, and it cannot be determined that the specific implementation of the present invention is limited to this; for technical personnel in the technical field to which the present invention belongs and related technical fields, based on the technical solution of the present invention, the expansion and replacement of operating methods and data should all fall within the protection scope of the present invention.

Claims

1. An experimental test method for estimating the bending strength and disturbance of rat femur, characterized in that: The method comprises the following steps: 1) Prepare a number of normal rats, and perform type 2 diabetes modeling on some of the rats to obtain a model group, then divide the modeled rats into three groups on average, and feed two of the groups with Chinese medicine reagents and Western medicine reagents respectively, and then obtain four groups of rat models with different blood glucose concentrations: normal group, model group, Chinese medicine group, and Western medicine group; 2) The femurs of the deceased rats in the four groups of rat models in step 1) were collected, the muscle tissues were removed, and then the femurs were placed in a phosphate buffered saline solution for culture treatment; 3) The rat femurs were removed from the phosphate buffered saline solution and dried, and then black and white speckles were sprayed on the upper surface of the femurs of the four groups of rats for digital image correlation processing; 4) The femurs of the four groups of rats were placed on a compression load loading device for three-point bending tests. A high-speed camera was used to take real-time photos of the fracture process of the femurs of the rats, and the test data were imported into a computer for processing; 5) deriving the rat femur load time history curve according to the test data obtained in step 4), and analyzing the displacement field and strain field between the fracture points of the rat femur; 6) Scan and analyze the rat femoral fracture surface using an electron microscope, input the scanned image information into Image J software for binarization processing, and after the conversion is completed, the diameter and area of ​​the rat femoral fracture surface can be accurately calculated based on the black and white pixel points of the binary image and the scale ratio of the electron microscope scanned image, and then the bending strength is calculated according to the bending strength theory formula of material mechanics, and then the bending strength and disturbance analysis are performed in combination with the image information of the high-speed camera; 7) Based on the analysis of the bending strength of the femurs of the four groups of rats, the biomechanical properties of the rat femurs were evaluated by mechanical strength.

2. An experimental test method for estimating the bending strength and disturbance of rat femur according to claim 1, characterized in that: In the step 1), the Chinese medicine reagent and the Western medicine reagent are respectively a Shenqi compound reagent and a Saxagliptin tablet reagent.

3. The experimental test method for estimating the bending strength and disturbance of rat femur according to claim 1, characterized in that: The specific analysis process in step 5) is as follows: a. Randomly select the points to be tested in the reference image of the rat femur P ( x i , y i ), and the point to be tested P ( x i , y i ) is used as the center to select a square sub-image with a size of (2N+1)×(2N+1) pixels, which is the reference subset; b. Use f ( x , y ) is represented; at the same time, from the deformed image, a square sub-image (M > N) with a size of (2M+1)×(2M+1) pixels is selected with the corresponding pixel point as the center, that is, the search subset; c. Select any deformation subset in the search subset and use g ′( x ′, y ′) indicates that the size of the deformed subset is (2N+1)×(2N+1) pixels; d. Then f ( x , y ) and g ′( x ′, y ′) Calculate the correlation according to the correlation coefficient formula to obtain the correlation coefficient distribution diagram of the whole field; e. The peak value of the correlation coefficient distribution graph is f ( x , y ) and g ′( x ′, y ′) is the best matching point; and the center point of the deformed subset P ′( x i ′, y i The difference between the coordinates of the center point of the reference subset is the point to be measured. P ( x i , y i )’s displacement vector; f. Using the same method, the above related operations are performed on each pixel point in the entire speckle pattern to obtain the full-field displacement and full-field strain of the rat femur, and then the displacement field and strain field of the rat femur can be further analyzed.

4. The experimental test method for estimating the bending strength and disturbance of rat femur according to claim 1, characterized in that: The compression load loading device in step 4) is an electro-hydraulic servo press equipped with a three-point bending fixture.

5. An experimental test method for estimating the bending strength and disturbance of rat femur according to claim 4, characterized in that: The three-point bending test in step 4) can be a central symmetrical loading or an eccentric asymmetrical three-point bending loading.

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