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

Abstract

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 70 Gy 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 2 mm 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.

Raman spectroscopy delineates radiation-induced injury and partial rescue by amifostine in bone: a murine mandibular model

“…The methods of animal selection, radiation and amifostine administration, recovery and care, and evaluation via Raman spectroscopy for the current 18 week control, XRT, and AMF-XRT specimens were identical to our prior study utilizing Raman spectroscopy to analyze 8 week control, XRT, and AMF-XRT specimens [15]. As such, findings and results were tabulated and compared between 8 and 18 week specimens.…”

“…We recently reported that the mineral compositions of irradiated rat hemi-mandibles, including mineral to matrix ratio and carbonate to mineral ratio and crystallinity, significantly differed from those of both control and AMF-XRT specimens at 8 weeks post-irradiation [15]. While 8 week AMF-XRT specimens showed mineralization patterns similar to controls, no difference was seen compared with 8 week XRT specimens in collagen cross-linking ratio.…”

“…Previous findings from our laboratory suggest that amifostine is capable of protecting bone against negative radiation-induced negative sequelae; we have also shown that Raman spectroscopy is a viable means by which to evaluate the chemical composition and ultrastructure of irradiated bone, having studied the effects of therapeutic-dose radiation in both the mandible and the tibia [15, 16]. However, as these two sites of bone are different in physiology and in biomechanical properties, our results are not directly transferable from one case to the other.…”

Total-body irradiation produces late degenerative joint damage in rats

“…Fixation of cartilage using 10% formalin has been shown to significantly lower T2 relaxation times versus unfixed controls (Fishbein et al 2007); the observed reduction in T2 relaxation times in fixed versus unfixed samples was in-part attributed to increased cross-linking. Abnormal cross-linking of collagen has previously been reported as a late radiation effect in skeletal tissues, specifically bone (Gong et al 2013; Tchanque-Fossuo et al 2013). We present T2 relaxation times from the 0 Gy control group that are similar to those in the literature (Figure 2) (Spandonis et al 2004).…”

Parathyroid hormone attenuates radiation-induced increases in collagen crosslink ratio at periosteal surfaces of mouse tibia

“…Previous work from our lab and others has shown that administration of amifostine, a radioprotectant, prior to irradiation results in reduction in free radical damage to growth plate function and in preservation of bone pre-irradiation mineralization patterns in irradiated animal models [8, 17, 32-34]. Anabolic agents, such as human recombinant PTH(1-34) peptide, have demonstrated capability to enhance bone regeneration processes, maintain more functional and heterogeneous collagen orientation, and increase bone mineral density via multiple processes.…”