Targeted Osteosarcoma Therapy via Tumor Treating Fields and MXene-Enhanced Scaffolds

Introduction: : Osteosarcoma is the most common type of primary bone cancer, affecting adolescents between the ages of 10-30 years old. Current standard treatment typically involves systemic chemotherapy combined with surgical tumor resection. Post-resection reconstruction requires bone grafting alongside radiation therapy (RT) to reduce the risk of recurrence. However, RT negatively impacts bone integrity, resulting in grafting failure. This highlights the need for alternative adjuvant therapies and strategies that limit tumor recurrence and enhance bone healing after tumor resection. Cryogels are macroporous, tissue-engineered scaffolds that have emerged as promising alternatives to bone grafts due to their ability to support osteogenesis. Tumor Treating Fields (TTFs) are a novel modality that apply low-intensity alternating electric fields (1–3 V at 100–300 kHz) to disrupt
mitotic spindle formation, inhibiting cancer cell proliferation. Currently FDA-approved for glioblastoma and mesothelioma, TTFs have shown complementary promise to RT and chemotherapy; however, their potential in osteosarcoma has yet to be explored. This is likely due to the inverse relationship between TTF efficacy and tissue conductivity [3], as bone inherently exhibits poor electrical conductivity. Recently, MXenes—2D ceramic nanomaterials composed of metal carbides, nitrides, or carbonitrides—have shown promise in tissue engineering due to their electrical conductivity, hydrophilicity, and tunable structure. By integrating MXenes into cryogel, we aim to create an electrically conductive, biomimetic scaffold capable of delivering TTFs to osteosarcoma cells. We hypothesize that MXene cryogel scaffolds will enable localized delivery of TTFs to the tumor site, thereby reducing osteosarcoma cell proliferation and migration while minimizing effects on healthy osteoblasts.

Materials and

Methods: : Cryogel scaffolds were prepared from a chitosan/gelatin (1:4) solution, and chemically crosslinked (1% glutaraldehyde) at -20°C. To incorporate MXene, a 70% Ti₃C₂Tₓ MXene ink was added to the solution; cryogels without MXene served as controls. Scaffolds were imaged via scanning electron microscopy and pore size analyzed by PoreVision software. Additional characterization included swelling, electrochemical impedance (EIS), and compression testing (N=3/group). Swell properties were tested in phosphate-buffered saline (PBS), measuring fluid absorption over a 24-hour period. To test mechanical strength, a compressive strain of 80% was applied to each sample. EIS was measured using a three-electrode setup in PBS, followed by cyclic voltammetry analysis. In vitro stimulation experiments used a custom-designed electrical stimulation chamber, adapted from Leppik et al. [2019], allowing simultaneous electrical stimulation of samples loaded in a 12-well plate. To test uniform chamber functionality, electrochemical degradation of methylene blue dye (3V for 48 hours) was measured spectroscopically. Biocompatibility was tested by seeding MG-63s for 7 days and analyzing via confocal imaging. Ongoing work is focused on seeding scaffolds with bone-marrow derived mesenchymal stem cells (BMSCs) or osteosarcoma cells (MG63s) and applying TTFs. As osteoblast cells are not
proliferative, they are expected to remain largely unaffected by TTFs, whereas TTFs will reduce the viability and proliferation of the MG-63s. Live/dead staining and MTS assay quantify viability and proliferation of all cells, both within control and MXene cryogels. A significant reduction in osteosarcoma cell viability/proliferation rates, relative to osteoblasts, will support the potential of TTFs for targeted inhibition of osteosarcoma progression.

Results, Conclusions, and Discussions:: While scanning electrom microscopy (SEM) images revealed similar visual appearance between cryogel groups (Figure 1A, B), analysis identified that MXene cryogels had significantly larger average pore size (2803.6 µm) compared to control (2098.9µm) (p < 0.05; Figure 2D). The larger pore size of the MXene cryogel is likely due to the more hydrophobic nature of MXene, which may play a beneficial role in cell infiltration, nutrient transport, and vascularization. There was no significant difference in absorption capability between cryogel groups determined by swell testing; similarly, compression testing showed no significant difference in compressive modulus between MXene and control cryogels (0.055 MPa vs. 0.050 MPa), demonstrating retained structural integrity for bone (p=0.05; data not shown). Cyclic voltammetry (CV) revealed MXene cryogels
exhibit a ~3.25x increase in conductivity, as evidenced by peak current densities in CV data (Figure 2E). Cell infiltration (MG63s) was achieved at 7 days in all cryogels, regardless of the presence of MXene (Figure 1C, D). Related to the custom-designed electrical stimulation chamber, the methylene blue degradation test confirmed uniform electrical stimulation across all 12 wells, with a variation of 9% between wells, indicating consistent stimulation conditions (Figure 2C). Ongoing work is focused on applying in vitro stimulation to the cellularized scaffolds to induce apoptosis through TTFs. Our preliminary results demonstrate that the incorporation of MXene retains the mechanical durability, swell characteristics, and cell viability of cryogel scaffolds. Functionality of our electrical stimulation chamber was demonstrated with methylene blue dye degradation in all 12 wells after 48 hours of stimulation. Further, the potential of cryogel scaffolds for bone regeneration is supported by our previous work showing increased bridging after resection in a murine model (Figure 3). This study establishes the feasibility of fabricating conductive scaffolds for TTF delivery, highlighting their potential to enhance adjuvant therapy for osteosarcomas. By combining electrical conductivity with biocompatibility, these scaffolds offer a promising alternative to radiation therapy (RT) following tumor resection—targeting cancer cells while preserving healthy bone tissue.

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