Behdod Poushanchi

Raman spectroscopy demonstrates Amifostine induced preservation of bone mineralization patterns in the irradiated murine mandible

PURPOSE Adjuvant radiotherapy in the management of head and neck cancer remains severely debilitating. Fortunately, newly developed agents aimed at decreasing radiation-induced damage have shown great promise. Amifostine (AMF) is a compound, which confers radio-protection to the exposed normal tissues, such as bone. Our intent is to utilize Raman spectroscopy to demonstrate how AMF preserves the mineral composition of the murine mandible following human equivalent radiation. METHODS Sprague Dawley rats were randomized into 3 experimental groups: control (n=5), XRT (n=5), and AMF-XRT (n=5). Both XRT and AMF groups underwent bioequivalent radiation of 70Gy in 5 fractions to the left hemimandible. AMF-XRT received Amifostine prior to radiation. Fifty-six days post-radiation, the hemimandibles were harvested, and Raman spectra were taken in the region of interest spanning 2mm behind the last molar. Bone mineral and matrix-specific Raman bands were analyzed using one-way ANOVA, with statistical significance at p<0.05. RESULTS The full-width at half-maximum of the primary phosphate band (FWHM) and the ratio of carbonate/phosphate intensities demonstrated significant differences between AMF-XRT versus XRT (p<0.01) and XRT versus control (p<0.01). There was no difference between AMF-XRT and control (p>0.05) in both Raman metrics. Computer-aided spectral subtraction further confirmed these results where AMF-XRT was spectrally similar to the control. Interestingly, the collagen cross-link ratio did not differ between XRT and AMF-XRT (p<0.01) but was significantly different from the control (p<0.01). CONCLUSION Our novel findings demonstrate that AMF prophylaxis maintains and protects bone mineral quality in the setting of radiation. Raman spectroscopy is an emerging and exceptionally attractive clinical translational technology to investigate and monitor both the destructive effects of radiation and the therapeutic remediation of AMF on the structural, physical and chemical qualities of bone.

Amifostine Remediates the Degenerative Effects of Radiation on the Mineralization Capacity of the Murine Mandible

Background: Radiotherapy, a cornerstone of head and neck cancer treatment, causes substantial morbidity to normal adjoining bone. The authors assessed the radioprotective effect of amifostine therapy on the mineralization of the mandible using micro–computed tomography. They hypothesized that amifostine would safeguard the mandible from radiation-induced disruption of the mineralization process and the associated failure of new bone creation. Methods: Male Sprague-Dawley rats were randomized into three groups: control (n = 8), radiation therapy (n = 5), and amifostine (n = 8). Animals in the radiation therapy and amifostine groups underwent human bioequivalent radiation of 70 Gy in five fractions to the left hemimandible. Fifty-six days after irradiation, the hemimandibles were harvested for radiomorphometric analyses. Results: Amifostine-treated animals exhibited less alopecia, mucositis, and weight loss in addition to increased cortical density in comparison with those treated with radiation therapy. Bone and tissue mineral densities showed statistically significant improvement in amifostine versus radiation therapy, and no difference was observed between amifostine and control groups. Detailed micro–computed tomographic analysis further demonstrated significant differences in the mineralization profile when comparing radiation therapy and amifostine. Amifostine maintained regions of lower mineralization consistent with the preservation of normal remodeling. Conclusions: The authors have successfully demonstrated the ability of amifostine pretreatment to protect the natural mineralization profile of bone. This reflects the capacity of amifostine prophylaxis to safeguard the normal surrounding mandible from the impediments of collateral damage imposed by irradiation. Further study can correlate these findings with the potential use of amifostine to prevent the devastating associated morbidities of radiotherapy such as pathologic fractures and osteoradionecrosis.

Amifostine protects vascularity and improves union in a model of irradiated mandibular fracture healing

Background: Pathologic fractures of the mandible can be devastating to cancer patients and are due in large part to the pernicious effects of irradiation on bone vascularity. The authors’ aim was to ascertain whether amifostine, a radioprotective drug, will preserve vascularity and improve bone healing in a murine model of irradiated mandibular fracture repair. Methods: Rats were randomized into three groups: nonirradiated fracture (n = 9), irradiation/fracture (n = 5), and amifostine/irradiation/fracture (n = 7). Animals in the irradiation groups underwent a human equivalent dose of radiation directed at the left hemimandible. Animals treated in the amifostine group received amifostine concomitantly with radiation. All animals underwent unilateral left mandibular osteotomy with external fixation set to a 2.1-mm fracture gap. Fracture healing was allowed for 40 days before perfusion with Microfil. Vascular radiomorphometrics were quantified with micro–computed tomography. Results: When compared with the irradiated/fractured group, amifostine treatment more than doubled the rate of fracture unions to 57 percent. Amifostine treatment also resulted in an increase in vessel number (123 percent; p < 0.05) and a corresponding decrease in vessel separation (55.5 percent; p < 0.05) there was no statistical difference in the vascularity metrics between the amifostine/irradiation/fracture group and the nonirradiated/fracture group. Conclusions: Amifostine prophylaxis during radiation maintains mandibular vascularity at levels observed in nonirradiated fracture specimens, corresponding to improved unions. These results set the stage for clinical exploration of this targeted therapy alone and in combination with other treatments, to mitigate the effects of irradiation on bone healing and fracture repair.

The effect of Amifostine prophylaxis on bone densitometry, biomechanical strength and union in mandibular pathologic fracture repair

BACKGROUND Pathologic fractures (Fx) of the mandibles are severely debilitating consequences of radiation (XRT) in the treatment of craniofacial malignancy. We have previously demonstrated Amifostines effect (AMF) in the remediation of radiation-induced cellular damage. We posit that AMF prophylaxis will preserve bone strength and drastically reverse radiotherapy-induced non-union in a murine mandibular model of pathologic fracture repair. MATERIALS AND METHODS Twenty-nine rats were randomized into 3 groups: Fx, XRT/Fx, and AMF/XRT/Fx. A fractionated human equivalent dose of radiation was delivered to the left hemimandibles of XRT/Fx and AMF/XRT/Fx. AMF/XRT/Fx was pre-treated with AMF. All groups underwent left mandibular osteotomy with external fixation and setting of a 2.1mm fracture gap post-operatively. Utilizing micro-computed tomography and biomechanical testing, the healed fracture was evaluated for strength. RESULTS All radiomorphometrics and biomechanical properties were significantly diminished in XRT/Fx compared to both Fx and AMF/XRT/Fx. No difference was demonstrated between Fx and AMF/XRT/Fx in both outcomes. CONCLUSION Our investigation establishes the significant and substantial capability of AMF prophylaxis to preserve and enhance bone union, quality and strength in the setting of human equivalent radiotherapy. Such novel discoveries establish the true potential to utilize pharmacotherapy to prevent and improve the treatment outcomes of radiation-induced late pathologic fractures.

Validation of Histologic Bone Analysis Following Microfil Vessel Perfusion.

Abstract The ability to examine bone vascularity using micro-computed tomography following vessel perfusion with Microfil® and to subsequently perform histologic bone analysis in the same specimen would provide an efficient method by which the vascular and cellular environment of bone can be examined simultaneously. The purpose of this report is to determine if the administration of Microfil precludes accurate histologic assessment of bone quality via osteocyte count and empty lacunae count. Sprague–Dawley rats (n = 6) underwent perfusion with Microfil. Left hemi-mandibles were harvested, decalcified, and underwent vascular analysis via micro-computed tomography prior to sectioning and staining with Gomori’s trichrome. Quantitative histomorphometric evaluation was performed. Ninety-five percent confidence intervals (CIs) were used to determine statistical differences from an established set of controls (n = 12). Histologic analyses were successfully performed on specimens that had been perfused. Quantitative measures of bone cellularity of perfused versus control specimens revealed no statistical difference in osteocyte count per high-power field (95·33 versus 94·66; 95% CI: −7·64 to 6·30) or empty lacunae per high-power field (2·73 versus 1·89; 95% CI: −1·81 to 0·13). A statistical validation is reported that allows histologic analysis of cell counts in specimens which had been perfused with Microfil.