Cryogel Scaffolds Induce Bone Formation following Femur Irradiation

Introduction: Bone is the third most common site for cancer metastasis, affecting ~66% of patients with common cancers—breast, lung, prostate, renal, thyroid—incurring skeletal events in up to 400,000 people in the US/year. Metastatic bone disease (MBD) results in weakened bone, leading to refractory pain and pathological fracture that increase disease state morbidity. Despite bone tissue’s dynamic nature and robust capability to remodel and regenerate following injury, MBD-affected bone does not heal according to normal principles, with average fracture healing rates for pathological fractures reported to be 8% at six months following injury. Further, the use of bone grafts for treatment offers additional complications including infection potential and donor site morbidity, where an improved treatment option is necessary. To combat this, cryogel scaffolds have been identified as ideal constructs to support bone formation following traumatic injury/disease. Preliminary work by our group has shown that cryogels do not lose their advantageous physical properties following radiation therapy (RT). Therefore, our overall premise is that MBD patients require alternative treatment options to i) improve bone formation and ii) accelerate healing. The central hypothesis of this study is that chitosan/gelatin cryogels will induce osseointegration and bone formation in MBD patients, while modulating tissue damage caused by RT. This hypothesis is based on current literature reviews, as well as previously published work by our group demonstrating cryogels and animal models for studying and improving bone formation. Our approach for creating a cost-effective, time-sensitive, and biologically improved targeted treatment option consists of optimized cryogel fabrication for MBD patients.

Materials and

Methods: Chitosan/gelatin (1:4) polymer in solution was poured into cylindrical molds (diameter 8mm), crosslinked with glutaraldehyde, and frozen at -20°C for 18 hours. Following freezing and subsequent thawing, cryogels were lyophilized for storage. Initial analysis included scanning electron microscopy (SEM), ultimate compression testing, swelling kinetics, and MG-63 (bone-like) cell infiltration. Following characterization, all scaffolds were sterilized with peracetic acid to prepare for in vivo analysis. Using an IACUC approved protocol, the femur of C57BL/6 skeletally mature mice (10-12 weeks old; male and female) were stabilized with a plate and a 3.5-mm long mid-diaphyseal femoral ostectomy was created using a Gigli saw under irrigation. Following defect creation, the defect site was either left empty or implanted with a cryogel scaffold. Following muscle and skin closure around the defect site, the mice immediately received clinically relevant doses of radiation (6-days x 6 Gray, 3-days x 10 Gray, or 1-day x 20 Gray), based on standard of practice/scale-down for a murine model. Mice were then monitored longitudinal via X-ray (radiographs) for 4 and 8 weeks from implantation to determine potential for new bone formation following RT exposure. Endpoint analysis included SEM/microCT to quantify bone formation (N=6/group/timepoint) and histology to determine healing and immune response (N=6/group/timepoint). Additionally, radiographs were scored for the degree of healing using a modified RUST scoring system. One-way ANOVA testing with Tukey post hoc was used to test significance (p < 0.05).

Results, Conclusions, and Discussions: Our overall objective is to analyze how cryogels respond to in vitro and in vivo treatment dosages of RT to optimize scaffold-induced osteogenesis in a long bone environment undergoing RT. We hypothesize that chitosan/gelatin cryogel scaffolds will improve osteogenesis in murine critical-sized defects during RT. While prior work has focused on cryogel scaffold-induced bone regeneration, until recently, no studies have tested cryogels in the presence of RT which could negatively affect the structure, function, and biocompatibility. Preliminary work by our group demonstrated no significant changes in modulus, regardless of RT dosage (assessed via SEM, compression testing, and swelling kinetics). However, cellular infiltration must be achieved to support new tissue growth. Previously published work by our group has demonstrated successful infiltration of osteosarcoma (MG-63) bone-like chitosan-gelatin cryogel scaffolds at timepoints over 28 days. Despite this, no work has evaluated the ability of cells to infiltrate scaffolding following exposure to RT, where RT in conjunction with- or post-surgery is common in MBD treatment regimes. Work by our group irradiated cryogel scaffolds and then seeded with osteosarcoma (MG-63) cells; despite exposure to RT, all cryogels exhibited full cell infiltration by 21 days (Fig. 1A.1, A.2). Following creation of the critical-size femur defect (Fig. 1B) and initial in vivo implantation, preliminary work has shown noticeable radiographic healing at the site of cryogel implantation. Ongoing work is focused on endpoint microCT and histological analysis. The outcomes include quantification of the osteogenic potential of cryogel constructs through mineral formation, proliferation, and bone tissue formation; we anticipate that this will demonstrate that cryogels support appropriate cellular infiltration, proliferation, matrix deposition, and mineral formation for bone healing in vivo, while in the presence of clinically relevant RT. Overall, the impact of cryogel scaffolds capable of inducing bone formation in the presence of RT has the potential to be highly translational to other clinical applications, including primary bone cancers.

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