Quantitative histologic evidence of amifostine-induced cytoprotection in an irradiated murine model of mandibular distraction osteogenesis

Abstract

Background: Head and neck cancer management requires adjuvant radiotherapy. The authors have previously demonstrated the damaging effect of a human equivalent dose of radiation on a murine mandibular model of distraction osteogenesis. Using quantitative histomorphometry, the authors' specific aim was to objectively measure amifostine radioprotection of the cellular integrity and tissue quality of an irradiated and distracted regenerate.

Methods: Sprague-Dawley rats were assigned randomly into two groups: radiotherapy/distraction osteogenesis and amifostine/radiotherapy/distraction osteogenesis, which received amifostine before radiotherapy. Both groups received a fractionated human equivalent dose of radiation prior to left mandibular osteotomy with fixator placement. Distraction to 5.1 mm was followed by a 28-day consolidation period. Quantitative histomorphometry was performed on left hemimandibles for osteocytes, empty lacunae, bone volume-to-tissue volume ratio, and osteoid volume-to-tissue volume ratio.

Results: Amifostine/radiotherapy/distraction osteogenesis exhibited bony bridging as opposed to radiotherapy/distraction osteogenesis fibrous unions. Quantitative histomorphometry analysis revealed statistically significant higher osteocyte count and bone volume-to-tissue volume ratio in amifostine-treated mandibles compared with irradiated mandibles. There was a corresponding decrease in empty lacunae and the ratio of osteoid volume-to-tissue volume between both groups.

Conclusions: The authors have successfully established the significant osseous cytoprotective and histoprotective capacity of amifostine for distraction osteogenesis in the face of radiotherapy. The amifostine-sparing effect on bone cellularity correlated with increased bony unions and elimination of fibrous union. The authors posit that the demonstration of similar efficacy of amifostine in the clinic may allow the successful implementation of distraction osteogenesis as a viable reconstructive option for head and neck cancer in the future.

Figure 1

Gross representative samples of left hemimandible from the radiated and distracted group, XRT/DO (left) and Amifostine pre-treated radiated and distracted group, AMF/XRT/DO group (right). Note the fibrous union exhibited in the region of the distracted gap (red arrow) of the XRT/DO specimen compared to the complete bony bridging observed in the AMF/XRT/DO regenerate. In addition, the XRT/DO specimen showed significant tissue loss over the coronoid process and the condyle (black arrow).

Figure 2

Coronoid section of the region of the distracted gap stained with Gomori Trichome. Non mineralized immature bone or osteoid highlighted in red by color thresholding was predominantly observed in the XRT/DO sample (red arrow), whereas mature bone highlighted in blue (black arrow) was abundant in the AMR/XRT/DO specimen.

Figure 3

Digital photograph under 16 × magnification further demonstrating differences between both groups. More empty lacunae (black arrows) and less mature osteocytes (red arrows) were consistently detected within the bone matrix in the XRT/DO group (left) compared to the AMF/XRT/DO group (right).

Figure 4

Graphs of the mean osteocyte count (left) and mean empty lacunae (right) per high power field. There were significantly more osteocytes and less empty lacunae in the AMF/XRT/DO group compared to the XRT/DO group (* denotes p<0.05).

Figure 5

Graphs of the percentage of mature bone (left) and immature bone (right) represented by the ratio of Bone Volume/Tissue Volume (BV/TV) and the ratio of Osteoid Volume/Tissue Volume (OV/TV) respectively. The AMF pre-treated specimens demonstrated a significant difference in mature bone as anticipated by an increase in the ratio of BV/TV and a resultant decrease in the percentage of immature bone compared to the XRT/DO specimens (* denotes p<0.05).