The Significant Impact of Fractionated Radiation Doses on Cryogel-Mediated Bone Regeneration in a Murine Femoral Critical Defect

Introduction: : Bone is a common site for cancer metastasis, affecting over 400,000 U.S. patients per year. Treatment for metastatic bone disease (MBD) typically includes radiation therapy (RT); however, conventional RT has been shown to adversely impact osteogenesis and fracture healing. Promoting bone regeneration in the presence of RT could greatly improve MBD treatment. Biomaterial scaffolds, including cryogels, promote cellular ingrowth and proliferation at bone defect sites. Cryogels are optimal for bone regeneration due to their favorable physical properties (i.e., pores, swelling, and durability), where studies by our group and others have demonstrated their ability to support osteogenesis both in vitro and in vivo. Additionally, previous work by this team has demonstrated that, while cryogel material properties exhibit significant changes that are dose dependent, initial data has suggested that cryogels that are hydrated at the time of radiation are protected from material alteration and remain effective at conducting cellular proliferation and adhesion in-vitro. However, no studies have explored the use of cryogels for bone regeneration in the context of radiation therapy in-vivo, particularly in elucidating their potential role in protecting/shielding healing bone from RT-induced damage. In this study we hypothesize that a chitosan/gelatin cryogel scaffold will conduct in-vivo osteogenesis in murine critical-sized femoral defects exposed to adjuvant doses of RT, regardless of the fractionated dosing treatment.

Materials and

Methods: : Chitosan/gelatin (1:4) in solution was crosslinked and frozen at -20°C for 18 hrs, thawed, and then lyophilized to create a cryogel scaffold. Following an approved IACUC protocol, 10–12-week-old skeletally mature C57BL/6 mice (n=36; male and female) underwent a 3-mm femoral critical-sized bone defect and were implanted with a cryogel scaffold before being randomly assigned to one of three RT treatment groups: 6 days x 6 Gray (n=12), 3 x 10 Gy (n=12), and 1 x 20 Gy (n=12). Treatment began immediately following surgery and continued for the designated time periods. Bone regeneration was monitored longitudinally via radiographs at one-week intervals. After four- or eight-weeks post-surgery, mice were sacrificed and harvested femurs were imaged via microCT and then prepared for paraffin histology, where sections were stained with H&E, Pircrosirius red, Alcian Blue, and Endomucin.

Results, Conclusions, and Discussions:: At 4 weeks, X-rays analysis reveal a median defect fill percentage close to 86% for all groups (Fig 1A, B). However, after 5 weeks of healing, mice in the 3x10 and 6x6 groups demonstrated significant bone resorption at the defect site compared to the 1x20 group. By 8 weeks, the 6x6 and 3x10 groups resorbed back to around 84% bone infill, significantly lower than the 1x20 group that reached 90% bone infill (Fig 1B; p< 0.05). MicroCT 3D reconstruction (Fig. 2A) and segmentation (Fig. 2B) revealed that while 6x6 defects show the most extensive peripheral mineral density deposition, the femur and defect site have resorbed and decreased in diameter and length, resulting in significant loss of bone volume across the defect compared to the 1x20 group, which exhibits more robust mineralization centrally. Quantification of growth reveals 1.345 mm³ for 1×20 Gy, 0.310 mm³ for 3x10 Gy, and 0.2967 mm³ for 6x6 Gy groups (Fig 2C). For the 6x6 group, histology sections depicted rounded cortical ends of the femur, with minimal integration with the cryogel centrally. However, there is evidence of significant cellular proliferation along the edges of the sample, including dense collagen type 1 deposition, blood vessel formation with red blood cells present (Fig. 4). The 1x20 group demonstrated more robust integration of the cryogel and native femoral bone at the defect edges, with deeper bone ingrowth toward the center of the sample, evidenced by cortical bone formation and mature blood vessels localized within the defect and cryogel core rather than at the periphery. (Fig. 4). Initial analysis suggests that low dose, prolonged radiation exposure alters the healing environment—potentially through changes in defect geometry and/or cell activity— promoting cell proliferation along the cryogel edges while accelerating resorption of the existing cortical interface. Ongoing work is evaluating cell and immune signaling during healing at empty femoral critical defect sites, with and without radiation, to evaluate the interplay between radiation and healing, and cryogel-mediated regeneration. Overall, this data supports a dose-dependent influence of radiation on the osteo-conductivity of cryogels and their integration with bone at defect sites following adjuvant radiation therapy.

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