(P 175) FIBRIN-ALGINATE INTERPENETRATING NETWORK HYDROGELS AS BIOMIMETIC PLATFORMS TO STUDY OSTEOSARCOMA

Objective: Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents. Despite its prevalence, the standard treatment has remained unchanged for 4 decades and offers less than a 25% 5-year survival rate for those with metastatic disease. This mandates a critical need to develop novel therapeutic targets for OS. This will only be possible once we have a better grasp of the initial progression and metastagenesis of this still poorly understood disease. Current OS in vitro models fail to account for important components of the bone marrow environment, including the three-dimensional (3D) nature of sarcomas, the physiological oxygen tension of bone marrow (5%), and the spatial and cellular heterogeneity of bone marrow. This complexity of OS and lack of improvement in treatment outcome necessitate the study of this disease in a novel system.

Hydrogels are effective platforms for use as biomimetic microenvironments for 3D cell culture. Fibrin and alginate are widely used to construct hydrogels due to many advantageous properties. To capitalize on the strengths of both materials, we developed fibrin-alginate interpenetrating network (FA IPN) hydrogels for simultaneous control over native adhesion motifs and tunable mechanical properties. We developed FA IPN hydrogels to investigate OS pathophysiology, specifically the interplay between oxygen tension, 3D vs monolayer culture, and co-culture with macrophages.

Methods: FA IPN hydrogels were formed with 3% w/v MVG sodium alginate, 2.5 µL/mL thrombin, 20 mg/mL fibrinogen, and crosslinked in 200 mM CaCl2 solution. Homotypic culture of highly metastatic K7M2 and less metastatic K12 osteosarcoma cells, as well as pre-osteoblastic MC3T3 control cells were entrapped in FA IPNs to form homotypic (HOM) cultures. Heterotypic (HET) culture was formulated by combining these cell lines with IC-21 macrophages at an 85:15 ratio. Cells were loaded at 1x106 cells/mL. Gels were cultured in complete media for up to 7 days in 5% and 21% oxygen (O2) to investigate the physiological relevance to native oxygen tensions in the bone compared to standard culture conditions. Cell proliferation was determined by total DNA content, but changes in individual cell type were not ascertained in this study.

Results: HOM proliferation increased in all cancerous groups over 7 days in culture in monolayer (Figure 1), but not 3D culture in FA IPNs (Figure 2). In HET cultures, macrophages inhibited K7M2 proliferation in 3D culture at 21% O2, but K12 proliferation was inhibited in all conditions except monolayer at 5% O2 (Figure 3). These data establish the impact of 3D culture, oxygen tension, and macrophage co-culture on proliferation as a first step toward tumorigenesis of OS.

Conclusion: Fibrin-alginate interpenetrating network hydrogels are a versatile and highly tunable platform. These studies demonstrate this biomaterial’s capacity to provide a biomimetic environment for the study of OS tumorigenesis.