Specimen preparation and treatment
To assess the effect of local adjuvants on cortical bone, fresh adult pig femurs were obtained from a certified slaughterhouse. All femur samples, including reference samples, were harvested from the right side of male pigs of the same age and weight. Specimens were derived from the midsection of the diaphysis, handled, and stored identically to minimize potential sources of bias. All specimens underwent the same thorough cleaning process and were subsequently stored for up to 1 week in a freezer at -19 °C.
A total of 35 samples were prepared, 7 for each type of local adjuvant, including reference samples. Samples were positioned into a piece of porcine muscle for better conductivity. Local adjuvants were applied to specimens under defined conditions:
1. Argon Beam Coagulation group treatment method
The argon beam coagulator was set at 120 W, as recommended for larger bones, and applied for exactly 30 seconds25. Then, after washing with saline, a second cycle of argon beam coagulation was performed. Next, the specimen was washed again by saline lavage.
2 Electrocautery group treatment method
The electrocautery was set at 100 W in spray mode and applied directly all over the cavity until the medular side of the diaphyseal surface was darkened6,24. Next, the specimen was washed by saline lavage.
3. Liquid Nitrogen group treatment method
Liquid nitrogen was sprayed out from the can toward the medular side of the diaphyseal specimens using a cryosurgical liquid nitrogen sprayer30,31. Once the ice on the surface of the cavity thawed, a second cryoablation cycle was performed.
4. Phenol group treatment method
Gauze pads soaked with 80% phenol were positioned on the medular side of the diaphyseal specimens for a period of 4 minutes6,32. Next, the specimen was washed by saline lavage. Two cycles of phenolisation were carried out.
5. Reference group treatment method
The reference samples were cleaned and stored exactly as the other samples, but no local adjuvant was applied. The samples were washed by saline lavage.
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the appropriate institutional review committee.
Micro-CT examination
To assess changes in bone mineral density (BMD), a specimen per type of local adjuvant was scanned. The specimens were scanned using a micro-computed tomography scanner (GE Phoenix v|tome|x L240, GE Sensing & Inspection Technologies GmbH, Wunstorf, Germany) with a voxel size of 14 μm. The bone density calibration was performed using a hydroxyapatite phantom (MicroCT-HA D20, © QRM GmbH, Moehrendorf, Germany) included during scanning. This phantom caliber consists of five cylinders with different densities – 0 mg/cm3, 50 mg/cm3, 200 mg/cm3, 800 mg/cm3, and 1200 mg/cm3. Micro-CT images of the phantom were then used to determine bone density according to a standard procedure based on a linear relationship between hydroxyapatite density and the pixel gray values of the corresponding image33,34. Due to quantitative analysis, all five samples, including the caliber, were scanned during one measurement.
A pixel gray value threshold of 10,000 is considered between hard tissue and air/soft tissues.
The pixel gray values of the volume of interest (VOI) were evaluated per specimen (Figure 1). The representative area was on the medular surface of the diaphyseal specimen and of a similar depth (0.2–0.4 mm). The pixel gray values of the volume were recorded and converted to BMD values. The examiner was blinded to the treatment type.
Mechanical examination
In the assessment of induced mechanical changes, the Vickers hardness test was employed. The Vickers hardness test is a commonly recognized method for measuring the hardness of a variety of materials, including cortical bone35. The Vickers hardness test uses a micro indentation with an indenter to the prepared specimen surface. The indenter has a square-based pyramid shape, and it is made of a very hard material36. This indenter was pressed to the medular surface of the specimen with the specified force for the indicated duration.
In this case, the applied load was 50 g for 10 s, using a Vickers hardness testing machine (INNOVATEST FALCON 800G2). The resulting indentation size is measured by high-magnification optics, and software developed for such a measurement evaluates the Vickers hardness (HV) based on the length of the two resulting diagonals of the indentation. The HV value (HV 0.05/10 s) represents the hardness of the material in the sense of resistance to deformation35. Higher HV values indicate harder materials. Three specimens per type were measured, and three indentations were made for each specimen, to ensure that the impact of the potential variation would be minimized. A total of nine indentations were made for each type of adjuvant and reference sample. The mean HV value per specimen was recorded, namely three mean values per type of adjuvant. The examiner was blinded to the treatment type.
Histological examination
To assess the induced depth of necrosis, tissue specimens were fixed in 10% neutral buffered formalin, embedded in paraffin, and routinely processed. Due to significant ossification of cortical bone, the material had to undergo decalcification in a solution of 8% hydrochloric acid with ferric chloride at room temperature. Decalcification was completed after 18 days37. Hematoxylin-eosin-stained tissue sections were evaluated using a Nikon microscope Eclipse Ci with a Nikon DS-Fi2 camera, and the depth of necrosis was measured using NIS-Elements D 4.13.04 software. The experienced pathologist was blinded to the treatment type.
Statistical analysis
The null hypothesis of this research is that the local application of an adjuvant does not influence the density and hardness of the bone. To test this hypothesis, statistical tests for comparing two unrelated samples were used, with one serving as the reference. The initial step involved the application of the Shapiro-Wilk statistic to assess the normality of the measured data. In cases where the data deviated significantly from a normal distribution, a nonparametric statistical test was preferred for analysis; otherwise, a parametric statistical test was applied. Based on the results of the Shapiro-Wilk test, the following tests were eventually used: Parametric two‐tailed t‐test was used to compare hardness test measurements. Nonparametric Mann-Whitney U test was used to compare bone density values for each type of treated specimen. A significance level of .001 was used for all statistical tests. Statistical analysis was performed using R software (version 4.0.5) in the RStudio development environment.