Mechanical and Histological Characteristics of Human Tubular Bones after Hyperthermal Treatment

Abstract

This research focused on studying regularities in changes in strength characteristics and histological patterns of healthy tubular bone tissue depending on the temperature setting of hyperthermal treatment. Experimentation has established that heating the experimental bone sample in a temperature range of 60 to 70 °C does not cause any decline in strength characteristics compared to the control samples not subject to heat treatment. In compression tests (along the length of the bone), after heating the bone samples ex vivo to 80 °C, the strength characteristics were found to increase as the samples sustained a higher maximum stress. In bending tests, in contrast, the strength characteristics were reliably found to decrease in bone samples at 80 °C and 90 °C for the maximum stress indicator and 90 °C for the modulus of elasticity. Data obtained through histological examination further demonstrated statistically significant differences between the two temperature ranges of 60–70 °C and 80–90 °C, where semi-quantitative assessment revealed statistically significant differences in the markers of bone tissue destruction caused by hyperthermal treatment. Moderate (at 60–70 °C) and pronounced (at 80–90 °C) dystrophic and necrotic changes were observed both in the cells and the intercellular matrix of the tibia. From a practical point of view, the temperature range of 60–70 °C can be considered operational for thermal ablation since, at these temperatures, no statistically significant decline was observed for the strength characteristics in either the cross-section or length-section.

1. Introduction

In the treatment of bone tumors, modern oncology gives preference to organ-preserving types of surgery that consist of both removal of the tumorous bone segment and the subsequent reconstruction of the affected bone [1,2]. Biomaterials used in reconstructive surgery are available in a rather wide range of artificial (metals, polymers, ceramics, etc.), natural (bone allotransplants, autotransplants), and composite osteoplastic materials [3,4,5,6].

Each of these materials has its advantages and disadvantages. At the same time, a patient’s own bone tissue remains a preferable choice, as it helps prevent any immune havoc or transmission of viral diseases (AIDS, etc.) and is biologically active and thus capable of initiating regenerative processes. It should be noted, however, that the maximum permissible volume of a patient’s autologous bone tissue is not always sufficient for reconstructive surgery. Techniques developed in order to address the above issues focus on the use of autotranplants after thermal treatment [7,8,9,10], where the tumorous bone area is removed and subject to thermal sterilization and is then repositioned to its original location in the body. A primary disadvantage of such a technique is poor osteo-integration (if any) of the thermally treated bone fragment with the mother bone and changes in its strength characteristics [11,12,13].

In order to eliminate the above deficiencies, Tomsk Cancer Research Institute has created an original technique for intraoperational high-temperature thermal ablation of tumorous bone tissues [14]. The originality of the new technique lies in its use of flexible surface heaters that encircle the tumorous bone segment and apply the heat in a specific temperature range. This technique does not require that the tumorous bone be surgically removed, which makes it possible to preserve the anatomical integrity of the bone and avoid the problem of poor osteo-integration of the thermally treated bone fragment with the mother bone. The technique is implemented using a Phoenix-2 local hyperthermy equipment package, which has been developed by the Tomsk State University of Control Systems and Radioelectronics [15,16,17,18]. The instrument is capable of applying a wide range of temperatures, thus allowing for various settings of thermal treatment.

In order to further develop the technique and introduce it into clinical practice, it is necessary to determine the optimal thermal conditions that would make it possible, on the one hand, to preserve the strength characteristics of bone tissue exposed to high-temperature treatment as much as possible, and on the other hand, to achieve a radical antitumor effect.

Our previous experiments on animal tubular bones revealed statistically significant changes in the strength properties of bones after the samples were heated to 100 °C, which indicated a critical ex vivo change in the biomechanical behavior of bone tissue at this temperature [19]. No statistically significant decline in strength characteristics was observed in the temperature range of 60–90 °C.

In this paper, we continued to study the strength characteristics of bone tissue, bringing the research area as close as possible to the target object of the technique. The experimental work was designed to validate and, if possible, supplement prior research and to identify the approximate temperature limits within which the strength characteristics of human tubular bones remain unaffected. The work was further supplemented with a histological study in order to analyze the changes that occur at the tissue level and compare the results with the data of mechanical testing of bone tissue.

2. Materials and Methods

Samples for the study were obtained from a patient with osteosarcoma of pelvic bones who, due to the pathological development of their condition, had to undergo amputation of the left lower limb. Samples for the study were obtained, with the patient’s consent, from their tibia that was not affected by the disease process, which was confirmed through X-ray and scintigraphy examinations.

The diaphyseal part of the bone was isolated and cut into 5 cylinders, each 4 cm long. The central cylinder was left intact and was referenced as the control sample. The cylinders were marked symmetrically in accordance with the temperatures applied to them (Figure 1 and Figure 2). The strain distribution is remarkably uniform along the human tibia under quasi-constant bending in the sagittal and frontal planes, under torsional and axial loading [20]. Therefore, a comparison of mechanical features of different parts of tibia diaphysis after hyperthermal treatment (Figure 1) was justified.

After the tibia was cut into cylinders, each cylinder was subjected to ex vivo thermal treatment at a specific temperature. The high-temperature treatment was applied in accordance with four temperature settings: 60 °C, 70 °C, 80 °C, and 90 °C. The duration of thermal treatment was 1 h. The heating was applied with the help of surface heaters of the Phoenix-2 local hyperthermy equipment complex; the temperature of the heaters was stabilized at a preselected level. A heat-insulating material was placed outside the heater to exclude the influence of the environment (Figure 3).

Temperature control involved Pt 100 thermal sensors placed on the bone surface (under the heater).

After high-temperature treatment, markings were drawn on all cylinders so that each cylinder was divided into 7 symmetric bars, 40 × 7 × 5 mm each, and the numbers designating the bars on all cylinders corresponded to each other (Figure 4 and Figure 5). E.g., bar 1 from the 60 °C cylinder corresponded to bars 1 from the 70 °C, 80 °C, and 90 °C cylinders and the control cylinder.

The bars were grouped in accordance with the study design:

–

Bars numbered 1, 2, 4, 5, and 6 were studied in bending experiments;

–

Bar 3 underwent a histological examination;

–

Bar 7 was studied in a compression experiment.

2.1. Compression Test Samples

For the purposes of the compression experiment, bars numbered 7 obtained from the five cylinders (4 of them from the cylinders after high-temperature treatment and 1 from the control cylinder) were further cut into fragments with dimensions 8 × 7 × 5 mm and marked. The samples were numbered in the same manner, from 1 to 5, from left to right (Figure 6).

As a result, 5 groups of 5 samples each were prepared (

Table 1. Groups of samples for compression test.

Group Number	Description	Quantity of Samples
1	60 °C	5
2	70 °C	5
3	80 °C	5
4	90 °C	5
5	Control	5

):

2.2. Bending Test Samples

Samples used for bending tests were bars with dimensions of 40 × 5 × 7 mm numbered 1, 2, 4, 5, and 6, prepared in the manner described above. For the test, 5 groups of 5 samples each were prepared (

Table 2. Groups of samples for bending test.

Group Number	Description	Quantity of Samples
1	60 °C	5
2	70 °C	5 (−1) *
3	80 °C	5
4	90 °C	5 (−1) *
5	Control	5

* As a result of some inaccuracy in the preparation of samples for bending tests, two bars (bar 1 from group 2 (70 °C) and bar 4 from group 3 (80 °C)) were found to be shorter than the others. These samples could not be used in bending tests since their length was less than the distance between supports (see Figure 9). They were eliminated from the experimental work.

).

2.3. Histological Study Samples

Histological (microscopic) examination of the condition of bar 3 from the human tibia that had been removed from the body and subjected to hyperthermal treatment ex vivo was carried out in accordance with the standard procedure [21].

The extracted implants were fixed for 24 h in 10% neutral formalin and decalcified for 7 days in a (1:1) mixture of 20% formic acid and 20% sodium acetate until the condition of the bone made it possible to make histological sections. The samples were then washed for 24 h to remove the decalcifying solution, placed for 24 h in 10% sodium sulfate, and then washed again with water for 24 h. Dehydration was achieved in eight changes of an isopropanol-based dehydrating solution in accordance with the manufacturer’s instructions (IsoPrep, Biovitrum, Saint-Petersburg, Russia), then the samples were placed in paraffin for impregnation. The samples were poured into a Histomix paraffin medium, and thin (5–7 microns) sections were made on the microtome perpendicular to the surface of the tissue laminae. The sections mounted on glass slides were stained with Gill’s hematoxylin and eosin for histological studies under standard conditions. The histological condition of bone tissue was assessed by means of microscopic examination of stained sections (using an Axioskop 40 microscope, Carl Zeiss, Goettingen, Germany), and digital images of the sections were taken (using a Power Shot A 630 camera, Canon, Tokyo, Japan; 14-megapixel resolution).

A semi-quantitative assessment (on a scale of 0 to 4 points) of cellular and tissue reaction of bone tissue to hyperthermal treatment was conducted in accordance with the recommendations of ISO 10993-6 [22]. The results of the microscopic reaction (by medians in each group) to the treatment were recorded as follows:

–

The treatment has no damaging effect: 0–0.9 points;

–

Light damage to the structural elements of bone tissue: 1–1.9 points;

–

Moderate damage: 2–2.9 points;

–

Pronounced damage: 3–3.9 points;

–

Severe damage: 4 points.

2.4. Compression Experiments

In order to obtain more accurate calculations, prior to the experiment, each of the selected bars was measured with a caliper in three mutually perpendicular dimensions: height, length, and width.

The mechanical strength of the samples was tested for compression using the Instron 1185 testing machine (ITW Inc., Glenview, IL, USA) [23,24,25]. For that purpose, each bar was placed between two parallel plates so that the long axis of the bone corresponded to the compression axis (Figure 7).

The compression rate was 1 mm/min. During the experiment, the Instron testing machine produced a set of force and strain values, which made it possible to calculate the necessary parameters and plot the graph of the stress–strain relationship. The test was stopped at the moment of critical strain of the sample, i.e., when a failure occurred. That moment was recorded as the maximum sustainable stress or stress-to-failure.

The stress value was calculated as follows (1):

$$\sigma =\frac{F}{S},$$

(1)

where

σ is the stress in MPa;

F is the force in N;

S is the area of the sample to which the pressure was applied in mm².

The strain was calculated relative to the initial height of the sample as follows (2):

$$\epsilon =\frac{\Delta h}{h},$$

(2)

where

ε is the unit strain, a non-dimensional value;

Δh is the change in the height of the sample in mm;

h is the initial height of the sample in mm.

Each value of stress, σ, uniquely corresponded to a value of strain, ε, which made it possible to plot the graph of stress/strain (σ/ε) dependency (Figure 8). Following that, the nature of the curve was evaluated, and a line was plotted as an approximation of its first steeply rising section that is linear in nature (line 1 in Figure 8). Then a point was found on the graph that corresponded to the deviation of the graph from line 1 by 0.2% (point σ_0.2 in Figure 8). A second line was plotted through that point, parallel to line 1 (line 2 in Figure 8). The point corresponding to the stress-to-failure value that the sample can sustain is indicated in Figure 8 as σ_max. The ordinate of this point corresponds to stress-to-failure, and the abscissa corresponds to strain-to-failure.

Another parameter that was determined based on the obtained data is the modulus of elasticity, which is calculated as the tangent of the inclination of line 1 or, given that the line can be defined by a y = kx + b type formula, the angle of inclination will correspond to the value of the coefficient k.

The difference between the abscissae of the point σ_0.2 and the point of intersection of line 1 with the axis of abscissae corresponds to elastic strain (ε_el), and the difference between the abscissae of points σ_max and σ_0.2 corresponds to plastic (elastic–plastic) strain (ε_pl).

2.5. Bending Experiments

The mechanical strength of the samples was tested for bending using the same Instron 1185 testing machine (ITW Inc., Glenview, IL, USA) with the same testing parameters that were used in the compression experiments. In this case, however, the action was applied to the center of the bar that was placed horizontally on two supports, periosteum up (Figure 9).

The speed of the pressure mechanism was 1 mm/min. During the experiment, the Instron testing machine produced a set of force and strain values, which made it possible to calculate the necessary parameters and plot the graph of the stress–strain relationship. The test was stopped at the moment of critical strain of the sample, i.e., when a failure occurred. That moment was recorded as the maximum sustainable stress or stress-to-failure.

The stress value was calculated as follows (3):

$$\sigma =\frac{\raisebox{1ex}{$\left(F · l\right)$}\!\left/ \!\raisebox{-1ex}{$4$}\right.}{\raisebox{1ex}{$\left(B · h^2\right)$}\!\left/ \!\raisebox{-1ex}{$6$}\right.},$$

(3)

where

σ is the stress in MPa;

F is the force in N;

l is the length of the sample in mm;

B is the width of the sample in mm;

h is the height of the sample in mm.

The strain was calculated relative to the initial height of the sample in accordance with Formula (2).

3. Results and Discussion

The data obtained in the compression and bending tests were statistically processed using the Statistica 10 software. The small sample size caused the results to be inconsistent with the normal distribution of characteristics (Shapiro–Wilk test) [26,27]. Therefore, the data were interpreted using non-parametric criteria and represented in the form of a median (Me) and a quartile range (Q1–Q3). The indicators were compared between the control group (before heating) and one of the groups after thermal treatment (60, 70, 80, and 90 °C). Parameters evaluated included stress-to-failure, strain-to-failure, elastic and plastic strain, and modulus of elasticity.

Samples for the compression tests were considered to be dependent since the groups were formed in a way that preserved the exact spatial correspondence of bars with the same numbers, reflecting their physiological location in the bone. Accordingly, the significance of any differences was assessed using the Wilcoxon test. Additionally, statistical differences were calculated using the Mann–Whitney test [28]. The purpose was to discuss the strength characteristics in this investigation with the data obtained at hyperthermia in the previous study, which was also ex vivo conducted on diaphysis of cadaveric porcine femurs [19]. Of course, pig bone biomechanics is not quite similar to human bone [29]. However, porcine bone is often used as a substitute for human bone in trauma studies because some biomechanical parameters are comparable to those reported for adult human bones [30]. Likewise, an understanding of hyperthermia in humans can be aided by a study of its similarities to the porcine model [31].

Statistically significant differences in the bending tests were identified using the Mann–Whitney test and the Kruskal–Wallis test. The use of non-parametric criteria was due to the different number of samples in the control group and the heat-treated groups (see Bending test samples). When processing histological data, non-parametric Mann–Whitney (P_U) and Wilcoxon (P_T) tests were applied for the independent and dependent samples, respectively.

3.1. Compression Test

3.1.1. Stress-to-Failure

When comparing the groups after hyperthermal treatment (60, 70, 80, and 90 °C) with the control group, an observation can be made that bone samples after hyperthermal treatment were able to sustain a higher compression stress-to-failure. Up to and including 80 °C, this indicator was shown to increase by 40% (P, Pu < 0.05; Figure S1); at 90 °C, this indicator decreased slightly (–5%) relative to the value of the control (non-heated) bone bar.

Similar results can be observed in the previous experiments with an animal cadaver bone [19]: after hyperthermal treatment in a range of 60–100 °C, bone tissue samples sustained a higher maximum compressive stress, and the greatest increase in the stress-to-failure indicator was observed in the 70 °C and 80 °C groups (by about 31–37%) with a statistical significance level of 0.052 and 0.064, respectively. Thus, while the previous experiment did not yield any significant difference (in accordance with the Mann–Whitney U test) between the control and the experimental groups (only potential trends were observed), this paper presents significant differences in the stress-to-failure indicator observed at 80 °C. A further increase in temperature results in the deterioration of bone structures, which manifests in a decrease in the maximum sustainable stress.

3.1.2. Strain-to-Failure

The next parameter for inter-group comparison was the strain-to-failure indicator; that is, the moment when the strain becomes a failure as a result of the applied stress was studied. Figure S2 details the change in strain-to-failure across groups.

The ex vivo experiment did not demonstrate a statistically significant effect of hyperthermia on the test indicator in the temperature range of 60–90 °C (Figure S2). A significant deterioration in bone behavior when strain-tested can develop at 100 °C, as demonstrated in our previous study on a healthy bone of a pig [19]. In view of this, the study also focused on the values of elastic and plastic strain (Figures S3 and S4).

3.1.3. Elastic Strain

A slight decrease in this indicator was observed in all groups after hyperthermal treatment compared to the control group, both in absolute numbers and in percentage (ranging from 13% to 37%). These values did not reach the level of statistical differences, however (Figure S3). A similar pattern was observed during the experiment with animal tubular bones [19].

3.1.4. Plastic Strain

By definition, plastic strain is the strain retained by the sample after the stress is relieved. It determines the failure limit (the mechanical stress at which the sample is destroyed).

In our experimental work, a certain decrease in the plastic strain value was observed at 60 °C, followed by an increase at temperatures of 70–90 °C (data are shown in Figure S4). However, the application of the Wilcoxon test revealed no statistically significant differences. The Mann–Whitney test similarly demonstrated no statistically significant differences. Nevertheless, it should be noted that at 90 °C, statistical significance reached the level of 0.06, which indicates a certain tendency toward growth of the indicator (by about 17% compared to the control group). As the degree of plastic strain increases, plastic properties may decrease so much that any further strain will cause failure. Similar results were obtained in experiments with a bone taken from a pig cadaver in the temperature range of 60–90 °C: a decrease at 60–70 °C and an increase at 80–90 °C. Again, a definitively significant increase in the plastic strain of a pig bone was achieved in the 100 °C thermal treatment group [19].

3.1.5. Modulus of Elasticity

The modulus of elasticity characterizes the ability of an object to become elastically deformed (i.e., to return to its original state after the effect of external forces is relieved). The ex vivo study (Figure S5) demonstrated an increase in the modulus of elasticity, while a statistically significant difference was observed at 60 °C and 70 °C.

At a higher temperature treatment (80 °C and 90 °C), the modulus of elasticity did not differ from the control value.

The compression study indicates that, within the test temperature range of 60–90 °C, ex vivo hyperthermia does not result in an excessive decline in the biochemical characteristics of the human tibia when subjected to a compression test. Furthermore, an increase in the modulus of elasticity (when heated to 60–70 °C) and stress-to-failure (at 80 °C) is observed, which indicates a certain “adaptation” of bone tissue to an extreme stimulus (stressor).

In terms of potential fundamental mechanisms underlying the above phenomena, it is possible to discuss the changes observed when protein is heated that manifest as a significant restructuring of macromolecules with a change in their tertiary and secondary structure due to the destruction of intramolecular bonds and hydrophobic interactions [32,33]. In its turn, the occurrence of hydrophobic radicals on the surface of molecules creates conditions that cause protein aggregation, which can ultimately contribute to an increase in bone density and cause an increase in the modulus of elasticity and maximum sustainable stress of the bone.

As the temperature increases further to 90 °C, thermal breakdown processes begin to prevail, with the destruction of peptide bonds and depolymerization of the fibrous matrix of the bone.

In order to confirm this hypothesis, a bending study of the biomechanical behavior of bone testing was conducted. This test makes it possible to study the elastic properties of bone tissue mainly associated with the state of the bone collagen matrix [34].

3.2. Bending Test

3.2.1. Stress-to-Failure

When comparing the groups by the level of sustained stress before failure, statistically significant results were obtained after hyperthermal treatment at temperatures of 80 °C and 90 °C. The maximum sustainable stress was observed to decrease (compared to the control group) by 33% and 24%, respectively (Figure S6).

The data indicates that the values of maximum sustainable stress after hyperthermal treatment vary depending on the point of application. In the compression test, an increase in this indicator was observed at a treatment temperature of 80 °C (Figure S1). In the bending test, it decreases when the sample is heated to 80 °C and 90 °C. In the course of interpretation, it can be assumed that at temperatures above 70 °C, the risk of a transverse fracture of the tibia increases.

3.2.2. Strain-to-Failure

The study of strain before failure in the bending test revealed fluctuations of values both downward at 60 °C and 70 °C (up to −23%) and upward at 80 °C and 90 °C (up to 15%) (Figure S7), although these changes were not statistically significant, similar to the compression test (Figure S2).

3.2.3. Elastic Strain

In contrast to compression tests (Figure S3), statistically significant differences were obtained in bending tests at a temperature of 90 °C in accordance with the Kruskal–Wallis test (p < 0.047) (Figure S8). The percentage increase in the indicator compared to the control group was about 81%.

3.2.4. Plastic (Plastic–Elastic) Strain

In general, there is a slight decline in this parameter of the tibia at any treatment temperature that does not reach the level of statistical difference from the control group (Figure S9).

3.2.5. Modulus of Elasticity

As shown in Figure S10, the modulus of elasticity in ex vivo hyperthermia of the tibia in the range of 60–90 °C progressively decreased by 2–71% compared to the control group. A rather pronounced decline in the modulus of elasticity (–52.37%) was observed after hyperthermal treatment at 80 °C, although these changes did not reach the level of statistical significance. A statistically significant decrease (of 71% compared to the control group, p < 0.05) of the indicator was achieved at a temperature of 90 °C.

Thus, in contrast to compression tests, when subjecting the human tibia to the bending test after ex vivo hyperthermal treatment, we observed a decline of biomechanical indices (maximum stress, modulus of elasticity). The temperature of 80 °C should be considered critical for healthy bone tissue; any further increase results in a sharp deterioration of its elastic properties.

3.3. Histological Data

Histological study showed that the bone tissue in the control group of samples (without hyperthermia) possessed a typical structure of long tubular bones without obvious signs of damage. At the same time, there was the presence of emptying osteocyte lacunae in the compact substance of the bone and sporadic bone lamina fragments in intertrabecular spaces of the spongy substance, which may be conditioned by sample preparation (Figure S11).

Histological examination after hyperthermia revealed same-type damage in the bone structure; its severity generally was proportional to the temperature applied (Figures S12 and S13). The task of identifying visual differences in these changes for each level of thermal treatment (60–70–80–90 °C) proved challenging since even the control (non-heated) samples demonstrated poor cellular composition, often with empty osteocyte lacunae.

Notable damage was observed when the bone was heated to over 80 °C (Figure S13). Therefore, it was possible to identify two temperature ranges of 60–70 °C (Figure S12) and 80–90 °C (Figure S13). In this case, a semi-quantitative assessment (in points) reveals statistical differences in the markers of bone tissue destruction caused by hyperthermal treatment (

Table 3. Histological assessment (tissue reaction, in points) of hyperthermic effect in various temperature ranges on the condition of human bones.

Structural Element of the Bone Tissue of Bar 3	60–70 °C		80–90 °C
	Description	Bone Condition Assessment, Points	Description	Bone Condition Assessment, Points
Compact substance	The bone matrix is uniformly mineralized, bone laminae attach tightly to one another, stratification of bone laminae is extremely rare (Figure S12)	1	Tissue samples become brittle and crumble while sliced. There are some areas with detaching bone laminae, mainly those adjacent to canals of osteons (Figure S13A)	2
Bone lacunae	Osteocyte lacunae are enlarged, often empty (Figure S12A,B)	2	Osteocyte lacunae are enlarged, mostly empty (Figure S13A–C)	3
Osteon canals	Often empty, some of osteon canals contain homogeneous necrotic mass, residual connective tissue and fragments of bone laminae (Figure S12A,B)	2	Empty or filled with homogeneous necrotic mass, residual connective tissue and fragments of bone laminae (Figure S13A,B)	3
Trabeculae of the spongy substance	Formed by mature lamellar bone tissue. Mostly, they tightly attached to each other. Sporadic areas of bone laminae disruption occur (Figure S12C)	2	Numerous areas of bone lamina disruption in the trabeculae in the spongy substance (Figure S13C)	3
Intertrabecular space of the medullary cavity	Often filled with homogeneous necrotic mass, residual connective tissue and fragments of bone laminae (Figure S12C)	2	Fragments of bone laminae are visible near the medullary cavity (Figure S13C). Soft tissues surrounding the bone with signs of necrosis with pronounced swelling of the intercellular matrix and destruction of collagen fibers (Figure S13D)	4
Me (Q1–Q3)	2.0 (2–2)		3.0 (3–3) PT = 0.043 PU = 0.012

Note: 0–0.9 points—no damaging effect; 1–1.9—light; 2–2.9—moderate; 3–3.9—pronounced; and 4—severe damages. Both Mann–Whitney (P_U) and Wilcoxon (P_T) tests were used to evaluate the differences between the bones treated with 60–70 °C or 80–90 °C.

). In accordance with ISO 10993-6 recommendations, moderate (at 60–70 °C) and pronounced (at 80–90 °C) dystrophic and necrotic changes were observed both in the cells and the intercellular matrix of human tibia (Table 3, Figures S12 and S13).

4. Conclusions

Exposure to high temperature changes the properties of bone tissue, and these changes have a different nature depending on the point of application of the external force. Our experiments made it possible to assess the nature and extent of these changes.

As a result of the research, the following was revealed:

• In compression tests, it was demonstrated that there was an increase in the sustainable stress-to-failure after the bone was heated ex vivo to 80 °C.
• In bending tests, it was demonstrated that a reliable decrease in the strength characteristics of the bone at 80 °C (stress-to-failure) and 90 °C (stress-to-failure, modulus of elasticity) occurs.
• The data of histological examination revealed statistically significant differences that characterize considerable damage to the bone tissue after hyperthermal treatment at the temperature of 80–90 °C.

Thus, from a practical point of view, the temperature range of 60–70 °C can be considered “operational” for thermal ablation since, at these temperatures, no statistically significant decline was observed for the strength characteristics in either the cross-section or length-section. These temperatures can be considered acceptable for clinical practice provided that they satisfy the additional condition of sufficiency for the destruction of malignant tumor cells and the possibility of full regeneration of healthy bone tissue after treatment. Therefore, further research in this area appears to have much promise for oncology. It is also necessary to trace and observe the stages of postsurgical regeneration of bone tissue and identify the time frame of prevalent osteolytic processes that will make the bone more sensitive to the usual stress, which may also require additional measures to strengthen the treated bone.