QUESTION/PURPOSE: Does a controlled deep-freezing temperature during irradiation help preserve the compressive mechanical properties of human femoral cortical bone allografts?

METHODS: Cortical bone cube samples, each measuring 64 mm3, were cut from the mid-diaphyseal midshaft of five fresh-frozen cadaver femurs (four male donors, mean [range] age at procurement 42 years [42 to 43]) and were allocated via block randomization into one of three experimental groups (with equal numbers of samples from each donor allocated into each group). Each experimental group consisted of 20 bone cube samples. Samples irradiated in dry ice were subjected to irradiation doses ranging from 26.7 kGy to 27.1 kGy (mean 26.9 kGy) at a deep-freezing temperature below -40°C (the recommended long-term storage temperature for allografts). Samples irradiated in gel ice underwent irradiation doses ranging from 26.2 kGy and 26.4 kGy (mean 26.3 kGy) in a freezing temperature range between -40°C and 0°C. Acting as controls, samples in a third group were not subjected to gamma irradiation. The mechanical properties (0.2% offset yield stress, ultimate compression stress, toughness, and the Young modulus) of samples from each group were subsequently evaluated via axial compression loading to failure along the long axis of the bone. The investigators were blinded to sample group during compression testing.

RESULTS: The mean ultimate compression stress (84 ± 27 MPa versus 119 ± 31 MPa, mean difference 35 [95% CI 9 to 60]; p = 0.005) and toughness (3622 ± 1720 kJ/m3 versus 5854 ± 2900 kJ/m3, mean difference 2232 [95% CI 70 to 4394]; p = 0.009) of samples irradiated at a higher temperature range (-40°C to 0°C) were lower than in those irradiated at deep-freezing temperatures (below -40°C). The mean 0.2% offset yield stress (73 ± 28 MPa versus 109 ± 38 MPa, mean difference 36 [95% CI 11 to 60]; p = 0.002) and ultimate compression stress (84 ± 27 MPa versus 128 ± 40 MPa, mean difference 44 [95% CI 17 to 69]; p < 0.001) of samples irradiated at a higher temperature range (-40°C to 0°C) were lower than the nonirradiated control group samples. The mean 0.2% offset yield stress (73 ± 28 MPa versus 101 ± 28 MPa, mean difference 28 [95% CI 3 to 52]; p = 0.02; effect size = 1.0 [95% CI 0.8 to 1.2]) of samples irradiated at higher temperature range (-40°C to 0°C) were no different with the numbers available to those irradiated at deep-freezing temperature. The mean toughness (3622 ± 1720 kJ/m3 versus 6231 ± 3410 kJ/m3, mean difference 2609 [95% CI 447 to 4771]; p = 0.02; effect size = 1.0 [95% CI 0.8 to 1.2]) of samples irradiated at higher temperature range (-40°C to 0°C) were no different with the numbers available to the non-irradiated control group samples. The mean 0.2% offset yield stress, ultimate compression stress, and toughness of samples irradiated in deep-freezing temperatures (below -40°C) were not different with the numbers available to the non-irradiated control group samples. The Young modulus was not different with the numbers available among the three groups.

CONCLUSION: In this study, maintenance of a deep-freezing temperature below -40°C, using dry ice as a cooling agent, consistently mitigated the adverse effects of irradiation on the monotonic-compression mechanical properties of human cortical bone tissue. Preserving the mechanical properties of a cortical allograft, when irradiated in a deep-freezing temperature, may have resulted from attenuation of the deleterious, indirect effects of gamma radiation on its collagen architecture in a frozen state. Immobilization of water molecules in this state prevents radiolysis and the subsequent generation of free radicals. This hypothesis was supported by an apparent loss of the protective effect when a range of higher freezing temperatures was used during irradiation.

OBJECTIVE: In this study, we report a rapid method for the residue analysis of IND and its metabolites, viz., IND-carboxylic acid, diaminotriazine, and triazine indanone in a wide range of palm oil matrices using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

METHOD: The optimized sample preparation workflows included two options: (1) acetonitrile extraction (QuEChERS workflow), followed by freezing at -80°C and (2) acetonitrile extraction, followed by cleanup through a C18 solid phase extraction (SPE) cartridge. The optimized LC runtime was 7 min. All these analytes were estimated by LC-MS/MS multiple reaction monitoring.

RESULTS: Both sample preparation methods provided similar method performance and acceptable results. The limit of quantification (LOQ) of IND, IND-carboxylic acid, and triazine indanone was 0.001 mg/kg. For diaminotriazine, the LOQ was 0.005 mg/kg. The method accuracy and precision complied with the SANTE/12682/2019 guidelines of analytical quality control.

CONCLUSIONS: The potentiality of the method lies in a high throughput analysis of IND and its metabolites in a single chromatographic run with high selectivity and sensitivity. Considering its fit-for-purpose performance, the method can be implemented in regulatory testing of IND residues in a wide range of palm oil matrices that are consumed and traded worldwide.

MATERIALS AND METHODS: A total of 24 ejaculates were collected from four bulls via an electroejaculator. Semen samples were diluted with 2% VCO in Tris-based extender which consists of various concentrations of SL (1, 1.25, 1.5, and 1.75%). A 20% egg yolk in Tris used as a positive control (C+). The diluted semen samples were divided into two fractions; one for chilling which were stored at 4°C for 24, 72, and 144 h before evaluated for semen quality parameters. The second fraction used for freezing was chilled for 3 h at 4°C, packed into 0.25 mL straws and then cryopreserved in liquid nitrogen. The samples were then evaluated after 7 and 14 days. Chilled and frozen semen samples were thawed at 37°C and assessed for general motility using computer-assisted semen analysis, viability, acrosome integrity and morphology (eosin-nigrosin stain), membrane integrity, and lipid peroxidation using thiobarbituric acid reaction test.

RESULTS: The results showed that all the quality parameters assessed were significantly (p<0.05) improved at 1.5% SL concentration in chilled semen. Treatment groups of 1, 1.25, 1.5, and 1.75% SL were higher in quality parameters than the control group (C+) in chilled semen. However, all the quality parameters in frozen-thawed semen were significantly higher in the C+ than the treated groups.