Investigating Human Cell Influence on the Micromechanical Environment of a 3D-Printed Bone Tissue Model

Abstract

Advanced stages of breast cancer (BC) are associated with the spread of tumors to distant sites, or metastasis, with the skeletal system being one of the most common locations for metastasis, with rates as high as 70%. Despite this, the mechanistic factors behind BC cell recruitment, infiltration, and survival in the bone niche remain underexplored. Extracellular vesicles (EVs) are membrane-bound particles that facilitate intercellular communication between cancer and bone cells to form pre-metastatic niches, infiltrate tissue systems, and promote survival. Current models, such as 2D in vitro cell culture and preclinical animal models, fail to replicate the physiological metastatic bone environment, leading to high failure rates of anti-cancer drugs and limited predictive accuracy. This study aims to enhance a previously developed 3D-printed bone tissue culture model to mimic the human bone microenvironment by seeding osteoblasts to assess metastatic mechanisms and the influence of breast cancer cell lines on in vitro model mechanical properties, in breast-to-bone metastasis. Dynamic mechanical analysis (DMA) was conducted on osteoblast-seeded scaffolds (n = 24) across 7-, 14-, and 21-day timepoints to assess changes in storage modulus, loss modulus, and damping coefficient (tanδ). SEM was used to evaluate cell adhesion, mineral deposition, and scaffold degradation, while μCT characterized porosity, strut thickness, and trabecular separation after long-term culture. To assess the metastatic influence of breast cancer extracellular vesicles (EVs), viability and proliferation assays were performed to optimize co-culture conditions and evaluate EV effects on osteoblast growth. Co-cultured scaffolds with human osteoblasts and T47D cells were analyzed by DMA at 4 and 7 days to assess mechanical changes, and osteocalcin ELISA was used to examine alterations in bone-specific protein expression. Voronoi scaffolds exhibited a decrease in stiffness over time, while TO scaffolds remained stable; Voronoi scaffolds maintained a significantly higher storage modulus (p = 0.038). SEM and μCT confirmed qualitative mineralization and degradation, along with scaffold print fidelity (i.e., Trabecular Separation = 521 µm), respectively. Co-cultured scaffolds showed a non-significant increase in both stiffness and osteocalcin expression. Together, these results support the use of this platform for modeling bone tissue in vitro as well as metastatic mechanisms in breast-to-bone cancer progression.