Conventional and FLASH Irradiated Cryogel Scaffolds for Bone Oncology

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 physical properties (i.e., pores, swelling, durability). It is unknown whether ionizing radiation affects the performance of cryogels. FLASH RT, a new form of RT that delivers ultrahigh-dose-rate radiation, demonstrates reduced tissue toxicity, and may therefore offer a benefit with cryogel use. We hypothesized that FLASH RT will not significantly affect cryogel physical properties, regardless of fractionated dose, whereas conventional RT will, when compared to each other and a control.

Materials and Methods:: Chitosan/gelatin (1:4) in solution was crosslinked and frozen at -20°C for 18 hours, then lyophilized. Cryogels were irradiated using either conventional external beam (X-Rad 320) or FLASH (Trilogy linac) irradiator systems; non-irradiated samples served as controls. Cryogels, hydrated in PBS or left dry, were exposed to 25, 39, and 48 Gray over 1-, 3-, or 6-day fractionated treatments, respectively. All scaffolds were imaged by SEM to quantify porosity, compressed to 50% displacement to assess modulus, and placed in water to determine swelling kinetics. Two-way ANOVA testing with Tukey, One-way ANOVA with Brown-Forsythe, and linear regression were used to test significance (P < 0.05).

Results, Conclusions, and Discussions:: Compression testing showed no significant changes in modulus for either RT treated samples when compared to control. FLASH samples (compression modulus figure) were significantly stiffer than conventionally irradiated hydrated samples at 1x25 Gray and dry samples at 3x13 Gray, receptively (P < 0.05). Swell testing indicated significant decreases in the swelling capacity of all conventional irradiated hydrated samples and 1x25 dry samples when compared to a control, whereas FLASH only significantly decreased in 6x8 hydrated group (P < 0.05). There were no significant differences in swelling capacity between conventional and FLASH samples for the same sample type (hydrated and dry) in each fractionated group (normalized swell capacity figure). Linear regression tests (fractionated dose vs mean swell capacity figure) showed that dry scaffolds exposed to conventional radiation significantly decreased in swelling as total fractionated dose increased (R² = 0.97), and hydrated scaffolds exposed to flash significantly increased in swelling capacity as total fractionated dose increased (R² = 0.52). Analysis of SEM images demonstrated that average pore diameter did not significantly change between hydrated or dry due to RT treatment. These results suggest swelling capacity and stiffness of both hydrated and dry cryogels are significantly affected by both the type of RT and fractionated dose. Both conventional and FLASH RT have never been used in combination with tissue-engineered scaffolds. MBD may benefit from cryogel treatment; however, the current results demonstrate that scaffold performance is affected by RT delivery and requires further study. Ongoing studies are focused on investigating cell infiltration/proliferation of osteoblast-like cells within the relevant RT groups, as well as the chemical modifications and degradation effects of RT exposure that induce the demonstrated physical property changes.

Acknowledgements (Optional): :

References (Optional): :