Abstract
We aimed to investigate the mechanical properties of canine long bones treated with liquid nitrogen for limb-sparing surgery (LSS) as described by Tsuchiya et al. Three-point bending tests were performed on left and right femurs and tibias obtained from healthy beagle cadavers to evaluate the bone mechanical properties. There were no significant differences in mechanical properties between the left and right bones from the same individual. Furthermore, none of the indices differed between liquid nitrogen-treated and untreated bones. Our findings suggest that liquid nitrogen treatment does not result in marked changes in the mechanical properties of canine long bones, and may provide basic mechanical information potentially relevant to LSS using liquid nitrogen-treated autografts.
Keywords: canine bone, limb-sparing surgery, liquid nitrogen, mechanical property, three-point bending test
Limb-sparing surgery (LSS) using liquid nitrogen-treated autograft (LNTA) for primary bone tumors, including osteosarcoma, in the long bones of the extremities is an established effective treatment in human medicine [7, 24]. Its advantages include simplicity of the procedure, excellent bone strength, preservation of bone morphogenetic proteins in the bone matrix, and cryoimmunization [23, 24]. A few case reports involved small animals have described LSS using LNTA for bone tumors in the appendicular skeleton [1, 5, 21, 29]. In LSS using LNTA, a tumor-containing portion of the long bone is surgically resected en bloc and decellularized ex vivo, followed by repositioning and internally fixing to the host bone using plates and screws. Next, the resected bone is exposed to the load generated by the patient’s ambulation. Therefore, the graft should maintain appropriate mechanical properties to allow transmission of the load of the affected limb. For large or obese dogs, these grafts are usually subjected to greater loads than normal, which may lead to fracture if the graft has poor mechanical properties [9]. In human medicine, the recommended method for cryopreserving tumor-affected bone fragments involves decellularizing the partially resected tumor-affected bone by freezing it in liquid nitrogen at −196°C for 20 min, followed by placement at room temperature (20°C) for 15 min and in saline at 30°C for 10 min [24]. This protocol is based on the results of compression tests on bovine metacarpal bones [28]. Additionally, the mechanical properties of bone following this freezing method have also been investigated [17]. However, these studies were conducted using the long bones of cattle and sheep, which significantly differ in size from those in dogs. Accordingly, there is a need to evaluate the effects of liquid nitrogen treatment on the mechanical properties of canine long bones. Therefore, we aimed to analyze the effects of liquid nitrogen treatment on the mechanical properties of canine long bones.
We collected 20 femurs and 20 tibias from the cadavers of 10 healthy beagle dogs that had been euthanized for unrelated reasons and stored at −20°C in a deep freezer (SRR-G681C; SANYO Electric Co., Ltd., Osaka, Japan). These dogs had been previously used in experiments approved by the Animal Ethics Committee of Nippon Veterinary and Life Science University (Approval numbers: 26S-43, 26J-27, 2019S-15, 2022K-77), from which the specimens used in the present study were obtained. The mean weight and age of the 10 dogs (two females, eight males) were 10.4 ± 1.5 kg and 21.3 ± 4.4 months, respectively (mean ± standard deviation [SD]). The median storage period in the freezer was 14.5 (range: 1–131) months. Individual characteristics and storage periods are shown in Table 1. The bones were thawed overnight at room temperature prior to the experiment, followed by length measurements. Femoral length was measured from the greater trochanter to the most distal point of the femoral condyle, while tibial length was measured from the intercondylar eminence to the medial condyle. Subsequently, the bones were prepared morphologically for three-point bending testing using a materials testing machine. Test specimens were prepared by resecting each bone perpendicular to the bone axis at 20% of their respective lengths from the proximal and distal ends. Next, the specimens and a bone mineral density (BMD) phantom (B-MAS200; Kyotokagaku, Kyoto, Japan) were simultaneously scanned using a computed tomography (CT) scanner (TSX-303A; TOSHIBA Corp., Tokyo, Japan) (slice width, 0.5 mm; slice interval, 0.5 mm; tube voltage, 120 kV; tube current, 300 mA). The cross-section of the diaphysis at the midpoint of the bone length was considered as a circle, with measurement of the outer and inner diameters of the cortical bone. Next, the ends of the specimens were fixed in dental resin (OSTRON II; GC Corp., Tokyo, Japan) to prepare the bone specimens for mechanical testing. The dental resin was fixed to form a 32-mm square, with its length extending to the inside of the support roll for the three-point bending test (Fig. 1A and 1B). Additionally, the BMD of each specimen was measured using quantitative CT [10]. A calibration curve was created using the CT values of the simultaneously assessed standard BMD phantom.
Table 1. Individual characteristics of the dogs used and storage period.
| Dog No. | Sex | Age (month) | Body weight (kg) | Storage period (month) |
|---|---|---|---|---|
| No.1 | Female | 20 | 8.4 | 131 |
| No.2 | Male | 23 | 9.9 | 56 |
| No.3 | Male | 25 | 10.0 | 55 |
| No.4 | Male | 14 | 9.1 | 24 |
| No.5 | Male | 25 | 10.4 | 5 |
| No.6 | Female | 16 | 9.0 | 127 |
| No.7 | Male | 25 | 12.9 | 3 |
| No.8 | Male | 24 | 11.0 | 3 |
| No.9 | Male | 25 | 12.6 | 3 |
| No.10 | Male | 16 | 11.0 | 1 |
| Mean ± SD / Median (range) | 21.3 ± 4.4 | 10.4 ± 1.5 | 14.5 (1–131) | |
Fig. 1.
Three-point bending test. For the femur (A) and the tibia (B), the load was applied at the cranial center of the shaft of the long bone, with a support span of 50 mm. After applying a preload of 50 N, a three-point bending test was conducted at a crosshead speed of 5 mm/min. The dental resin was fixed up to the length of the inner surface of the cylinder of the lower jig (dotted line).
In Experiment 1, we compared the mechanical properties of left and right long bones obtained from the same individual. Here, five bilateral pairs of femurs and tibias (20 bones) from the same individuals (Dogs No. 1 to 5, Table 1) underwent mechanical testing without liquid nitrogen treatment. In Experiment 2, we investigated the effects of liquid nitrogen treatment on the mechanical properties of long bones. Specifically, we performed mechanical testing of right-sided and left-sided bones with and without liquid nitrogen treatment, respectively. The bones treated with liquid nitrogen were designated as the liquid nitrogen group (LN group), and those without treatment were designated as the control group. This experiment involved five bilateral pairs of femurs and tibias (20 bones) from the same individual (Dogs No. 6 to 10, Table 1). Liquid nitrogen treatment was performed as described by Tsuchiya et al. Specifically, the bones were immersed in liquid nitrogen for 20 min, left at room temperature for 15 min at 20°C, and immersed in saline at 30°C for 10 min [24]. Subsequently, the specimens underwent BMD measurements as well as three-point bending tests using a universal testing machine (AUTOGRAPH AGS-X; SHIMADZU, Kyoto, Japan). The test was conducted with supporting points (L) of 50 mm, with the load being applied on to the center of the bone diaphysis from the anterior side until break. Bones may exhibit mechanical anisotropy, resulting in direction-dependent mechanical properties [14]. Therefore, in this study, three-point bending tests were performed in the cranial direction, as commonly described in previous studies [11, 15]. After applying a preload of 50 N, the three-point bending test was performed at a crosshead speed of 5 mm/min, followed by calculation of load-displacement curves. During the specimen preparation and testing, Ringer’s solution was sprayed on the test bones at 5-min intervals to keep them moisturized. We determined the maximum load, fracture load, displacement at maximum load, displacement at fracture, and stiffness from the load-displacement curves. Additionally, the area moment of inertia (I) of each specimen, which was calculated from the outer and inner cortical bone diameters on CT, was used to determine the maximum stress, fracture stress, bending stiffness, and elastic modulus. Specifically, I was calculated as I=π × (outer diameter 4 −inner diameter 4) / 64. Stiffness was calculated as the slope from 20% to 80% of the fracture load based on the load-displacement curve [27]. The elastic modulus (E) was calculated as E=stiffness × L3 / (48 × I). The bending stiffness was calculated as bending stiffness=E ×I.
The normality of data distribution was assessed using the Shapiro-Wilk test. Normally and non-normally distributed variables are presented as mean ± SD and median (range), respectively, with between-group comparisons using a paired t-test and Wilcoxon signed-rank test, respectively. For side-to-side comparisons, differences were calculated as right−left (LN group−control group), and the mean differences with 95% confidence intervals (CIs) were reported. Multiple comparisons across parameters were adjusted using the Holm–Bonferroni method. In Experiment 2, repeated-measures analysis of variance was used to compare the BMD among the pre- and post-treatment liquid nitrogen (LN-pre BMD and LN-post BMD) groups and the control group (Cont BMD). To assess whether storage duration influenced the treatment effect of liquid nitrogen, the association between storage period and treatment-related side-to-side differences (LN group−control group) in mechanical parameters was evaluated using Spearman’s rank correlation as an exploratory analysis. All statistical analyzes were performed using standard statistical software (Stata version 14.0; StataCorp, College Station, TX, USA), and statistical significance was set at P<0.05.
Tables 2, 3, and 4 present comparisons of mechanical parameters, mean differences with 95% CIs, and BMD between the left and right femurs and tibias of the same animals in Experiment 1. There were no significant differences in mechanical parameters and BMD between the left and right bones from the same individual. Tables 5, 6, and 7 present comparisons of mechanical parameters, mean differences with 95% CIs, and BMD between femurs and tibias in the same animals in the LN group and the control group in Experiment 2. In Experiment 2, no difference was observed in the mechanical parameters of the femurs and tibias between the LN group and the control group (Tables 5 and 6). Furthermore, there were no significant differences in BMD among the three groups (cont BMD, LN-pre BMD, and LN-post BMD) for both the femur and tibia (Table 7). The results of the correlation analysis between storage duration and liquid nitrogen-induced changes in mechanical parameters are shown in Table 8. Significant correlations were observed for I and maximum stress in the femur, and for maximum load and displacement at maximum load in the tibia.
Table 2. Comparison of mechanical properties between the left and right femurs.
| Femoral side | Mean ± SD / Median (range) | Mean difference (95% CI) | P-value (unadjusted) | P-value (holm-adjusted) | |
|---|---|---|---|---|---|
| Maximum load (N) | Right | 1,652.4 ± 166.7 | 31.2 (−61.5 to 123.8) | 0.403 | 1.000 |
| Left | 1,621.2 ± 205.8 | ||||
| Fracture load (N) | Right | 1,583.3 ± 181.2 | 88.0 (−298.5 to 474.6) | 0.562 | 1.000 |
| Left | 1,495.3 ± 299.8 | ||||
| Displacement at maximum load (mm) | Right | 2.7 ± 0.6 | 0.28 (−0.56 to 1.12) | 0.410 | 1.000 |
| Left | 2.4 ± 0.1 | ||||
| Displacement at fracture (mm) | Right | 3.4 ± 0.6 | 0.11 (−0.88 to 1.11) | 0.770 | 1.000 |
| Left | 3.3 ± 1.0 | ||||
| Stiffness (N/mm) | Right | 814.6 ± 237.0 | −93.8 (−433.8 to 246.1) | 0.486 | 1.000 |
| Left | 908.4 ± 133.5 | ||||
| Area moment of inertia (mm2) | Right | 701.3 ± 218.9 | −51.3 (−170.8 to 68.2) | 0.299 | 1.000 |
| Left | 752.7 ± 274.9 | ||||
| Maximum stress (MPa) | Right | 164.5 ± 54.6 | −1.40 (−22.8 to 20.0) | 0.865 | 1.000 |
| Left | 165.9 ± 49.8 | ||||
| Fracture stress (MPa) | Right | 158.0 ± 55.4 | 1.15 (−20.5 to 22.8) | 0.890 | 1.000 |
| Left | 156.8 ± 61.8 | ||||
| Bending stiffness (×106 N/mm) | Right | 2.1 ± 0.7 | −0.24 (−1.13 to 0.64) | 0.486 | 1.000 |
| Left | 2.4 ± 0.4 | ||||
| Elastic modulus (GPa) | Right | 3.3 ± 2.0 | −0.18 (−1.46 to 1.09) | 0.708 | 1.000 |
| Left | 3.5 ± 1.4 |
Mean differences and 95% CIs were calculated for the right–left sides. CI: confidence interval.
Table 3. Comparison of mechanical properties between the left and right tibias.
| Tibial side | Mean ± SD / Median (range) | Mean difference (95% CI) | P-value (unadjusted) | P-value (holm-adjusted) | |
|---|---|---|---|---|---|
| Maximum load (N) | Right | 1,674.6 ± 270.1 | 19.7 (−75.8 to 115.1) | 0.598 | 1.000 |
| Left | 1,655.0 ± 209.7 | ||||
| Fracture load (N) | Right | 1,357.4 ± 206.5 | −140.5 (−531.3 to 250.3) | 0.375 | 1.000 |
| Left | 1,497.9 ± 259.8 | ||||
| Displacement at maximum load (mm) | Right | 2.0 ± 0.1 | −0.08 (−0.51 to 0.35) | 0.624 | 1.000 |
| Left | 2.1 ± 0.4 | ||||
| Displacement at fracture (mm) | Right | 3.5 ± 1.0 | 0.17 (−2.44 to 2.78) | 0.865 | 1.000 |
| Left | 3.3 ± 1.0 | ||||
| Stiffness (N/mm) | Right | 1,190.0 ± 178.5 | 22.0 (−150.3 to 194.3) | 0.741 | 1.000 |
| Left | 1,168.1 ± 69.8 | ||||
| Area moment of inertia (mm2) | Right | 727.9 ± 144.8 | −24.7 (−196.7 to 147.3) | 0.710 | 1.000 |
| Left | 752.0 ± 190.1 | ||||
| Maximum stress (MPa) | Right | 146.1 (132.6–237.5) | 4.62 (−23.7 to 33.0) | 0.405 | 1.000 |
| Left | 159.1 (109.7–202.1) | ||||
| Fracture stress (MPa) | Right | 131.3 ± 20.8 | −14.5 (−55.9 to 26.9) | 0.385 | 1.000 |
| Left | 145.8 ± 37.4 | ||||
| Bending stiffness (×106 N/mm) | Right | 3.1 ± 0.5 | 0.06 (−0.39 to 0.51) | 0.741 | 1.000 |
| Left | 3.0 ± 0.2 | ||||
| Elastic modulus (GPa) | Right | 4.4 ± 0.9 | 0.11 (−1.02 to 1.24) | 0.800 | 1.000 |
| Left | 4.3 ± 0.9 |
Mean differences and 95% CIs were calculated for the right−left sides. CI: confidence interval.
Table 4. Comparison of bone mineral density between the left and right femurs and tibias.
| Side | BMD | P-value | |
|---|---|---|---|
| Mean ± SD (mg/cm3) | |||
| Femur | Right | 915.0 ± 99.5 | 0.083 |
| Left | 865.9 ± 36.3 | ||
| Tibia | Right | 945.6 ± 87.9 | 0.138 |
| Left | 913.0 ± 56.2 |
BMD: bone mineral density.
Table 5. Comparison of mechanical properties of the femur between the liquid nitrogen and control groups.
| Femoral side | Mean ± SD / Median (range) | Mean difference (95% CI) | P-value (unadjusted) | P-value (holm-adjusted) | |
|---|---|---|---|---|---|
| Maximum load (N) | LN group | 1,480.8 ± 266.4 | −165.7 (−373.5 to 42.1) | 0.091 | 0.819 |
| Control group | 1,646.6 ± 268.2 | ||||
| Fracture load (N) | LN group | 1,441.3 ± 320.0 | −133.6 (−247.6 to −19.5) | 0.031 | 0.310 |
| Control group | 1,574.9 ± 279.4 | ||||
| Displacement at maximum load (mm) | LN group | 2.2 ± 0.7 | −0.16 (−1.77 to 1.44) | 0.792 | 1.000 |
| Control group | 2.3 ± 0.6 | ||||
| Displacement at fracture (mm) | LN group | 2.4 ± 0.8 | −0.43 (−2.52 to 1.67) | 0.600 | 1.000 |
| Control group | 2.8 ± 0.9 | ||||
| Stiffness (N/mm) | LN group | 934.2 ± 210.8 | −44.8 (−380.9 to 291.2) | 0.726 | 1.000 |
| Control group | 979.0 ± 128.9 | ||||
| Area moment of inertia (mm2) | LN group | 749.6 ± 270.0 | −83.5 (−204.4 to 37.4) | 0.128 | 1.000 |
| Control group | 833.1 ± 287.5 | ||||
| Maximum stress (MPa) | LN group | 146.0 ± 26.6 | −7.9 (−50.3 to 34.4) | 0.631 | 1.000 |
| Control group | 154.0 ± 48.9 | ||||
| Fracture stress (MPa) | LN group | 141.3 ± 29.6 | −4.7 (−32.2 to 22.8) | 0.66 | 1.000 |
| Control group | 146.0 ± 43.4 | ||||
| Bending stiffness (×106 N/mm) | LN group | 2.4 ± 0.5 | −0.12 (−0.99 to 0.76) | 0.730 | 1.000 |
| Control group | 2.5 ± 0.3 | ||||
| Elastic modulus (GPa) | LN group | 3.6 ± 1.5 | 0.29 (−0.90 to 1.48) | 0.533 | 1.000 |
| Control group | 3.3 ± 1.0 |
Mean differences and 95% CIs were calculated for the LN group −Control group. LN group: liquid nitrogen group; CI: confidence interval.
Table 6. Comparison of mechanical properties of the tibia between the liquid nitrogen and control groups.
| Tibial side | Mean ± SD / Median (range) | Mean difference (95% CI) | P-value (unadjusted) | P-value (holm-adjusted) | |
|---|---|---|---|---|---|
| Maximum load (N) | LN group | 1,702.7 ± 250.7 | −54.2 (−163.5 to 55.2) | 0.241 | 1.000 |
| Control group | 1,756.9 ± 270.5 | ||||
| Fracture load (N) | LN group | 1,375.2 ± 322.9 | −32.2 (−258.2 to 193.8) | 0.713 | 1.000 |
| Control group | 1,407.4 ± 389.7 | ||||
| Displacement at maximum load (mm) | LN group | 1.9 ± 0.2 | −0.13 (−0.32 to 0.06) | 0.131 | 1.000 |
| Control group | 2.0 ± 0.1 | ||||
| Displacement at fracture (mm) | LN group | 4.1 ± 0.8 | 0.22 (−0.50 to 0.95) | 0.444 | 1.000 |
| Control group | 3.8 ± 1.0 | ||||
| Stiffness (N/mm) | LN group | 1,291.0 ± 94.5 | 19.0 (−24.3 to 62.3) | 0.289 | 1.000 |
| Control group | 1,271.9 ± 97.5 | ||||
| Area moment of inertia (mm2) | LN group | 705.5 ± 194.2 | −100.4 (−213.1 to 12.3) | 0.069 | 0.690 |
| Control group | 805.9 ± 197.9 | ||||
| Maximum stress (MPa) | LN group | 171.1 ± 16.2 | 13.9 (−10.9 to 38.6) | 0.195 | 1.000 |
| Control group | 157.3 ± 21.4 | ||||
| Fracture stress (MPa) | LN group | 137.4 ± 23.9 | 9.5 (−15.7 to 34.7) | 0.355 | 1.000 |
| Control group | 127.9 ± 39.3 | ||||
| Bending stiffness (×106 N/mm) | LN group | 3.4 ± 0.2 | 0.05 (−0.06 to 0.16) | 0.289 | 1.000 |
| Control group | 3.3 ± 0.3 | ||||
| Elastic modulus (GPa) | LN group | 4.3 (4.0–6.5) | 0.79 (−0.07 to 1.65) | 0.080 | 0.720 |
| Control group | 4.3 (3.3–5.6) |
Mean differences and 95% CIs were calculated for the LN group −Control group. LN group: liquid nitrogen group; CI: confidence interval.
Table 7. Comparison of bone mineral density between the liquid nitrogen (LN-pre, LN-post) and control groups.
| Cont BMD | LN-pre BMD | LN-post BMD | P-value | |
|---|---|---|---|---|
| Mean ± SD (mg/cm3) | Mean ± SD (mg/cm3) | Mean ± SD (mg/cm3) | ||
| Femur | 889.7 ± 47.8 | 890.7 ± 94.9 | 875.7 ± 75.5 | 0.971 |
| Tibia | 873.1 ± 66.2 | 890.4 ± 59.5 | 906.9 ± 70.8 | 0.893 |
BMD: bone mineral density; Cont BMD: bone mineral density of the control group; LN-pre BMD: bone mineral density before liquid nitrogen treatment; LN-post BMD: bone mineral density after liquid nitrogen treatment.
Table 8. Spearman’s rank correlations between preservation period and liquid nitrogen-induced changes in mechanical parameters of the femurs and tibias.
| Femur | Tibia | |||
|---|---|---|---|---|
| ρ | P-value | ρ | P-value | |
| Maximum load | 0.67 | 0.22 | 0.89 | 0.04 |
| Fracture load | 0.22 | 0.72 | 0.45 | 0.45 |
| Displacement at maximum load | 0.45 | 0.45 | 0.89 | 0.04 |
| Displacement at fracture | 0.67 | 0.22 | −0.22 | 0.72 |
| Stiffness | −0.22 | 0.72 | 0.67 | 0.22 |
| Area moment of inertia | −0.89 | 0.04 | −0.22 | 0.72 |
| Maximum stress | 0.89 | 0.04 | 0.45 | 0.45 |
| Fracture stress | 0.67 | 0.22 | 0.22 | 0.72 |
| Bending stiffness | −0.22 | 0.72 | 0.67 | 0.22 |
| Elastic modulus | 0.22 | 0.72 | 0.22 | 0.72 |
The association between the preservation period and the differences in mechanical properties between the liquid nitrogen and control groups was evaluated using Spearman’s correlation coefficient. ρ; Spearman’s rank correlation coefficients.
In our study, we observed no significant differences in the bone mechanical properties between the left and right bones from the same healthy dog. These findings were largely consistent with previous results from three-point bending tests of canine femurs and tibias [15]. A three-point bending test of beagle tibias reported a maximum load of 929.4 ± 69.6 N, stiffness of 595.9 ± 65.4 N/mm, maximum stress of 159.3 ± 8.1 MPa, elastic modulus of 6.1 ± 0.4 GPa, and bending stiffness of 2.7 ± 0.3 × 106 N/mm [18]. Another study reported a maximum load of 1,047.6–1,243.8 N, a maximum stress of 209.5–289.8 MPa, an elastic modulus of 3.9–8.0 GPa, and a bending stiffness of 1.8–2.4 × 106 N/mm [6]. Maximum load and bending stiffness reflect the bone structural properties; however, they are easily affected by bone shape and experimental conditions, which result in variability. Contrastingly, stress and elastic modulus, which reflect the bone material properties, are consistent. Generally, our values for stress and elastic modulus are comparable to those reported in previous studies [20]. After confirming the absence of significant left–right differences in mechanical properties, we compared the mechanical properties of liquid nitrogen-treated and intact bones in Experiment 2 using paired femurs and tibias from the same individuals. In Experiment 2, no significant differences were observed in the mechanical parameters of the femur and tibia between the LN group and the control group. Previous studies have evaluated the maximum load, compressive load, compressive stress, and compressive strain of bovine and ovine bones treated with liquid nitrogen using compression and four-point bending tests. These studies reported no strength differences between liquid nitrogen-treated and untreated bones, consistent with the results from the present study [17, 28]. In addition, these previous studies reported that, although the differences were not statistically significant, liquid nitrogen treatment tended to reduce bone strength compared with that of normal bone. Although the liquid nitrogen treatment methods differed from those used in the present study, previous reports have shown that liquid nitrogen treatment can induce microcracks in the bone [12] and that freezing at −70°C can cause collagen degeneration [2]. In our study, the mechanical indices tended to be lower in the LN group than in the control group; however, there were no significant differences. Histological evaluation of the tested bones was not performed; however, these factors may have contributed to the observed decreases in several mechanical parameters following liquid nitrogen treatment in both the femur and the tibia. Considering these findings, liquid nitrogen treatment may affect mechanical properties, but the extent of these effects is likely limited. Additionally, a study reported no significant difference in crack displacement between rabbit bones treated with liquid nitrogen and those frozen at −80°C [13]. In the present study, there was no significant between-group difference in displacement at fracture. It has also been reported that, under freezing conditions different from those used in the present study, the elastic modulus may be either higher or lower than that of untreated bone [4, 25]. Moreover, although the freezing conditions differed, previous studies have suggested that freezing may exert minor effects on hydroxyapatite and on the organic components of the extracellular matrix [8, 16]. Although previous studies employed different protocols, they reported only slight changes in BMD following liquid nitrogen treatment [3]. Consistent with these findings, no statistically significant changes in BMD were observed in the present study. Taken together, our findings regarding mechanical properties and BMD are largely consistent with those of previous reports, and the results of the present study indicate that liquid nitrogen treatment did not significantly impair bone strength. However, liquid nitrogen treatment may weaken the mechanical properties of osteosarcoma-affected bones compared to normal bones [22]. Depending on the degree of weakening, additional treatments such as bone graft or bone cement reinforcement may be necessary [24].
In Experiment 2, the right side was assigned to the LN group, and the left side served as the control group. Previous studies have reported no marked left–right differences in the mechanical properties of canine long bones [15]. In addition, in Experiment 1, we confirmed that there were no significant differences in mechanical parameters between the left and right sides of the same individual. Based on these findings, treatment was consistently applied to the right side to maintain a uniform definition of paired comparisons. However, with this experimental design, the potential influence of laterality could not be fully eliminated. The behavior of mechanical parameters following liquid nitrogen treatment in bones with long preservation periods remains unclear. In the present study, liquid nitrogen treatment was applied to specimens that included bones with extended preservation durations; therefore, the relationship between preservation period and treatment-related changes in mechanical parameters was assessed in an exploratory manner. In the femur, I showed a significant negative correlation, whereas maximum stress showed a significant positive correlation with the preservation period. One possible explanation is that prolonged storage may have increased bone fragility, and subsequent liquid nitrogen treatment could have induced microstructural alterations. Even small changes in cross-sectional geometry may substantially affect I, given its dependence on the fourth power of the radius, which in turn may influence calculated stress values. In the tibia, maximum load and displacement at maximum load each showed significant positive correlations with the preservation period. Although freezing may affect the organic components of the extracellular matrix and is generally expected to reduce mechanical parameters [8], a positive correlation was observed for maximum load in the present study, indicating an opposite trend. Furthermore, the observed correlations were not consistent across parameters or between the femur and tibia. This lack of consistency may be attributable to the limited sample size, as well as the fact that only a single specimen had an extended preservation period. In addition, although this exploratory analysis involved multiple comparisons, no adjustment for multiple testing was applied; therefore, the results should therefore be interpreted with caution. Consequently, further studies using a larger number of specimens are required to fully elucidate the relationship between preservation duration and changes in mechanical parameters following liquid nitrogen treatment.
This study has several limitations. First, the collected specimens were stored for varying periods. Bone storage at −20°C for 8–12 months has been shown not to affect mechanical properties [19, 26]. To minimize the influence of storage duration on the findings, we compared mechanical properties between the left and right bones of the same animal. In Experiment 2, the relationship between storage period and the effects of liquid nitrogen treatment was also analyzed in an exploratory manner. Nonetheless, the influence of differences in storage duration could not be completely ruled out. Second, although no statistically significant differences were observed in the measured mechanical parameters, this does not necessarily indicate that the treated bones exhibit mechanical strength equivalent to that of normal bone. Indeed, previous studies have reported a tendency toward reduced mechanical strength compared with that of normal bone [17, 28]. Third, we evaluated the bone mechanical properties under bending, but not rotational or compressive loads. Therefore, further studies applying different loading conditions are warranted to elucidate the mechanical properties of liquid nitrogen-treated long bones. Fourth, the sample size was relatively small (n=5 bilateral pairs), which may limit the statistical power to detect differences between the liquid nitrogen-treated and control bones. Therefore, the absence of statistically significant differences, particularly in Experiment 2, does not necessarily indicate the absence of a true difference, and the possibility of a type II error should be considered.
In conclusion, under the loading conditions examined in this study, no statistically significant deterioration in the mechanical properties of canine long bones was detected following liquid nitrogen treatment. These findings suggest that liquid nitrogen treatment does not markedly alter mechanical properties relevant to LSS using LNTA. However, in clinical applications, it is necessary to consider the effects of bone tumors on osseous tissue, and because long bones are subjected to multidirectional mechanical loading, further studies using different loading modes and larger sample sizes are warranted to characterize more fully the mechanical behavior of liquid nitrogen-treated long bones.
CONFLICT OF INTEREST
The authors have nothing to disclose.
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