Experimental study on deantigenization and trabecular structure effects on bovine cancellous bone compression

Samples and preparation

Fresh bovine femur bones, slaughtered on the same day, were procured from a local market. After removing the periosteum and surrounding soft tissues, the bones were immediately stored in a −35 °C freezer for 24 h. While frozen, the femurs were sectioned, focusing on areas with a high concentration of cancellous bone. Cylindrical samples were extracted from the mid-region of the femoral head using a hollow drill with an inner diameter of 5 mm. A total of 25 cylindrical samples were randomly punched from the cancellous bone region above the bone scale line, ensuring uniform sampling by avoiding the ligamentous fossa at the femoral head's apex. Post-punching, the samples were trimmed and polished to achieve cylindrical dimensions of Ø4 × 8 mm (Figure 1).

Degreasing and deproteinization

Due to slight variations in the mechanical properties of femoral head cancellous bone based on positional distribution, and to mitigate the influence of cortical bone effects, the 25 cylindrical samples were randomly divided into five groups (A, B, C, D and E), each containing five samples. Group A served as the control group and remained untreated.

Methanol has polarity and penetration-enhancing effects, and together with chloroform's strong non-polar solubility, it can efficiently extract lipids, 1:1 volume ratio mixture of methanol and chloroform is a well-known and classic method for degreasing. 30% H₂O₂ solution, as a strong oxidizing agent, can oxidize and degrade bone collagen, while also destroying other organic components. It is a commonly used deproteinization method in the field of biochemistry.

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Figure 1.

Samples preparation steps.

Group B samples were degreased by soaking in methanol/ chloroform solution for 24 h. Group C samples were degreased by immersion in a 30% H₂O₂ solution for 24 h, with the solution replaced every 12 h. Group D samples underwent a combined treatment: first, they were degreased by soaking in a 1:1 methanol /chloroform solution for 24 h, followed by deproteinization in a 30% H₂O₂ solution for two 12-h intervals, with the solution refreshed after the first 12 h. Group E samples were treated in the reverse order of Group D, with deproteinization first followed by degreasing (Figure 2).

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Figure 2.

Samples de-antigenization steps.

After the respective chemical treatments, all samples were immersed in anhydrous ethanol for 10 h to remove residual reagents and surface antigens.

Structural characterization of cancellous bone

Micro-CT scanning, a nondestructive 3D imaging technique, utilizes X-rays to irradiate samples and records the intensity distribution of transmitted X-rays via a detector. This process enables the reconstruction of high-resolution 3D internal structural images using computer algorithms, making it particularly suitable for imaging and analyzing fine and intricate structures. In this experiment, five groups of samples were placed separately on the stage (nano voxel-3000D Sanying Precision Instruments), and the scanner performed imaging scans at a resolution of 500 nm, an exposure time of 0.4 s per projection, an energy of 90 kV, and a current of 66.5 ± 0.2 µA. Reconstruction was performed using the instrument's proprietary software (New Recon). The resulting slices were exported for analysis, and parameters such as trabecular bone volume fraction, average trabecular thickness, trabecular anisotropy, and fractal dimension were calculated using specialized software plugins.

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Figure 4.

Fractal dimension box-counting method evaluation schematic. ¹⁵

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Figure 5.

Slicing effect after fitting calculation of bone trabecular thickness and trabecular spacing. (a) binarized slice, (b) trabecular thickness, (c) trabecular spacing.

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Figure 6.

Electronic universal testing machine and loading process of compression experiment.

To minimize the impact of surface densification artifacts introduced during sample preparation, the region of interest (ROI) was carefully selected within the scanned volume using the software. A cylindrical region, one size smaller than the original sample and centered within the cylinder, was defined as the ROI for export and subsequent analysis. This approach ensured the accuracy and reliability of the structural parameter measurements.

Hardness test

The cylindrical specimens were thawed at room temperature for 2 h while immersed in physiological saline, the lower end of each sample was fixed. A Shore hardness tester was employed to measure the hardness of the samples. To minimize measurement error, hardness values were recorded at five distinct positions on each sample: upper left, lower left, upper right, lower right, and the center. The highest and lowest values were excluded, and the average of the remaining three measurements was calculated and recorded as the hardness value for that sample.

Compressive strength test

Compression tests were conducted on cancellous bone samples subjected to different de-antigenization treatments. Before testing, the samples were thawed at room temperature for 2 h. The experiments were performed using a universal electronic testing machine (Hegewald & Peschke, Inspekt Table Blue 5 kN) with two kinds of load capacity, 500 N and 5 KN, for this test 500 N load capacity will be used. Displacement-controlled loading was applied at a rate of 1 mm/min, and a preload of approximately 0.5 N was uniformly applied at the onset of loading.Due to the small cross-sectional area of the sample, no lubrication is applied between the sample and the fixture. The tests were carried out at room temperature (23 ± 1 °C, 50 ± 5%RH), with the samples subjected to axial strain up to 25% (Figure 6). Throughout the loading process, the testing machine collected real-time data, including time, load, and displacement. The load-displacement curve was plotted using Origin software based on the load and displacement data obtained from the universal testing machine during the experiment.

σ s = F A

(1)

The stress values corresponding to each data point on the force-displacement curve can be calculated.

ε = δ L 0

(2)

The strain values corresponding to each data point were calculated. By plotting the stress and strain values of all data points as a line graph, the stress-strain curve was obtained.

Young's modulus describes a material's ability to resist elastic deformation. Given the pronounced viscoelasticity of cancellous bone, Young's modulus serves as a critical parameter for characterizing its mechanical properties. This value is obtained by calculating the slope of the linear region in the stress-strain curve. The yield strength is defined as the stress at which a material begins to deform plastically, it corresponds to the peak stress at which plastic deformation initiates on the stress-strain curve. Compressive strength represents the maximum stress a material can endure under compressive loading. For certain elastoplastic materials, yield strength may serve as a reasonable approximation of compressive strength. Given the discernible yield point observed in the experimental stress-strain curve, yield strength was employed to characterize the compressive strength of trabecular bone.

Statistical analysis

One-way analysis of variance was used on the obtained hardness data to determine the significance of the correlation between different de-antigenization methods and treatment sequences with bone hardness differences, and then Tukey's test was selected for multiple post-hoc comparisons based on the results of the homogeneity of variance assumption. One-way ANOVA was applied to yield strength and Young's modulus, with ANOVA or Welch's tests performed based on the reported homogeneity of variance. Bivariate correlation analysis was conducted between the computed bone structural parameters and compressive performance parameters, and parameters with significant effects were selected for further multiple linear regression analysis based on the Pearson's and corresponding p-values.

Cancellous bone hardness

The hardness values of five groups are presented in the table below:

Pairwise comparisons across five sample groups, including de-antigenization versus controls, are presented in Table 1 (P -values and confidence intervals). Results indicated that defatting alone did not significantly alter bone hardness (P = 0.843). In contrast, deproteinization significantly reduced material hardness. Furthermore, a synergistic effect was observed when defatting followed deproteinization (Group B/E, P = 0.032), yielding a greater softening effect compared to single or reverse-ordered treatments. No additional hardness reduction occurred when defatting preceded deproteinization (Group C/D, P = 1.000). The effect size η 2 = S S b e t w e e n S S t o t a l  = 0.539 was large, which falls within the range of a large effect in biomedicine. These findings demonstrate that de-antigenization exerts both statistically and clinically meaningful effects on bone material hardness.

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Table 1.

Sample hardness of the five ABCDE groups.

Group	Average hardness value (HB)	Mean difference	P	95% confidence interval
A (Control Group)	63.750 ± 4.802	-	-	-
B (Degreased Group)	60.333 ± 4.446	3.417	0.843	−6.592,13.426
C (Deproteinized Group)	53.633 ± 6.607	10.117	0.047	0.108–20.126
D (Degreased & Deproteinized Group)	53.599 ± 5.728	10.151	0.046	0.142–20.160
E (Deproteinized & Degreased Group)	49.600 ± 4.535	14.150	0.003	4.141–24.159

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Table 2.

Yield strength and Young's modulus of five groups.

Group	Yield strength ( σ s , MPa)	Average Young's modulus (E,GPa)
Control Group (A)	27.346 ± 2.257	0.564 ± 0.252
Degreased Group (B)	24.552 ± 6.769	0.397 ± 0.047
Deproteinized Group (C)	25.615 ± 6.595	0.362 ± 0.061
Degreased & Deproteinized Group (D)	20.240 ± 6.590	0.350 ± 0.104
Deproteinized & Degreased Group (E)	17.561 ± 8.507	0.263 ± 0.136

Effect of deantigenization on the hardness of cancellous bone

A comparison between groups A and B indicates that the degreasing treatment of cancellous bone using a methanol /chloroform solution resulted in merely a 5% reduction in sample hardness, compared with other control groups, its effect on sample hardness was not statistically significant (Table 1). In comparisons among groups A and C, after immersion in H₂O₂ solution, the cancellous bone specimens exhibited a 15.87% reduction in hardness, demonstrating that deproteinization treatment significantly compromises the mechanical integrity of trabecular tissue. Comparing groups C and D, it was found that performing defatting treatment before deproteinization does not increase the degree of reduction in hardness, but comparing Group D and Group E, it was found that deproteinization before defatting the damage to the hardness of the trabeculae.

Therefore, when employing H₂O₂ solution for degreasing cancellous bone, it is crucial to control the processing time and solution concentration to avoid excessive reduction in sample hardness, which could compromise the mechanical properties of the bone. Simultaneously, when deproteinization and degreasing of the bone are required, degreasing should be performed first to minimize damage to the bone's hardness.

Effect of deantigenization on the compressive strength of cancellous bone

Based on the compression experiment data obtained from the universal testing machine generated compression load-displacement curves, and strength-strain curves for each group. The compression curves of bovine cancellous bone exhibited an overall trend of initial increase, followed by a decrease, and then a continuous rise, with a distinct elastic-plastic phase observed in the material's behavior (Figure 7).

The comparisons between groups A/B and A/C respectively revealed that the degreasing treatment reduced the yield strength of cancellous bone by approximately 10.2% and 6.3%. The comparisons between groups A/D and A/E respectively revealed that the degreasing treatment reduced the yield strength of cancellous bone by approximately 26.0% and 35.8%. Although statistical analysis did not detect significant differences among groups (P = 0.513), potentially due to limited statistical power, descriptive trends suggest that there is still a trend similar to the changes observed in hardness. At the same time, statistical analysis indicates that yield strength exhibits a significantly similar trend to that of the Young's modulus.

Effect of structural parameters of bone trabeculae on compressive strength of cancellous bone

The results indicate that the yield strength of trabecular bone is significantly negatively correlated with Tb.Th and significantly positively correlated with FD (Figure 11), the corresponding trend can also be seen in Figure 8(c) and (e). Although DA is simply positively correlated with yield strength, its effect in the regression model is overshadowed by Tb.Th and FD (or due to their shared influence).

Based on the data obtained from Figure 11, a multiple regression equation for yield strength was formulated:

σ s = − 86.843 − 15.012 T b . T h + 8.556 D A + 39.862 F D

Since the Young's modulus is highly correlated with the yield strength (P < 0.01, r = 0.713), its trend is considered to be consistent with that of the yield strength. These findings highlight the importance of trabecular structural parameters in determining the mechanical properties of cancellous bone, providing valuable insights for optimizing bone scaffold design and clinical applications.