Poster S7 - Developing a 3D In Vitro Bone Niche for Evaluating the Interactions of Osteoblasts and Metastatic Breast Cancer Cells

Introduction: Fundamentally, a model approximates a physical or biological system, a "biomimicking" model will have enough richness around the selected features to provide useful information about a biological system. Generally, models have the potential to provide control of inputs, access to the states (internal conditions in the model), and measurement of the outputs to support new understanding and predictions of the physical system. Osteoclasts can be directly and/or indirectly activated by breast cancer cells, inducing the progression of breast cancer bone metastasis (BCBM) through growth factor-triggered tumor growth-bone absorption-tumor growth.1 However, a lingering question in biomedicine probes the role of osteoblasts in the homing and growth of metastatic breast cancer. Indeed, as clinical investigations are challenging, in vitro models are a potential tool to investigate the interplay between the bone microenvironment and breast tumor cells leading to bone metastases. Therefore, we seek to build a “biomimicking” in vitro 3D bone niche that mimics the mineral component and the porous geometry of native bone. The bone model will be used to explore the interactions between breast cancer cells and pre-osteoblasts of the bone metastatic niche under healthy and osteoporotic bone conditions in order to unlock new knowledge for preventing and treating BCBM.

Materials and

Methods: A composite of 45wt% polylactide (PL) and 55wt% nano-β-tricalcium phosphate (nTCP) was prepared2 and then extruded into a 1.75mm (nominal) diameter filament at 165°C. The filament was used in standard 3D printers to create scaffolds with variable geometries, including an osteoporotic niche model with semi-gyroid pore shape (0.5BD denoting 50% bone density (BD)) and a healthy bone niche with 0.6BD (7×5 mm in diameter × height). The melting point of the TCP composite, filament diameter, surface roughness, and pore geometry (n ≥3) were measured. Subsequently, 1×105 MC3T3 pre-osteoblast cells in 75µL media were seeded onto the scaffolds and cultured for 7 days followed by dynamic seeding of 1×105 MCF-7 breast cancer cells. Scaffolds with no pre-seeded MC3T3 cells served as controls. The cell adhesion was analyzed (MC3T3: 7 days after seeding; MCF-7: 1 hour after seeding) by alamarBlueTM assay and quantified as metabolism percent reduction. Differences in all the quantifiable results were assessed by Student’s t-test and one-way ANOVA with pairwise comparison.

Results, Conclusions, and Discussions: No difference in melting point was observed (Fig.1A), suggesting that the 153°C melting point was stable. Prior to 3D printing, the composite was extruded as filament through a screw-driven filament extruder. Filaments from separate batches (Fig.1B) had similar diameters (1.62 ± 0.1mm), suggesting good reproducibility during the controllable (temperature and speed) extrusion process. Filaments also presented varied surfaces (Fig.1C); the surface roughness of the TCP composite filament (10.8 ± 1.36µm) was significantly higher than the PL filament (2.51 ± 0.31µm) likely due to of the additive mineral phase. Stereoscope analyses (Fig.1D) demonstrated that the average pore size of the 0.5BD osteoporotic niche (830 ± 13.5µm) was significantly higher than that of the 0.6BD healthy niche (546.5 ± 30.1µm), supporting the precise and consistent manufacturing of the 3D niche with complex geometries and controllable pore sizes to mimic bone healthy and osteoporotic conditions. As shown in Fig.2, the TCP groups generally yielded higher cell adhesion than PL groups. Similarly, cell adhesion in 0.6 BD groups was higher than that in the 0.5BD groups. TCP 0.6 BD supported the highest number of MC3T3 and MCF-7 cells (0.85 ± 0.056 and 0.11 ± 0.012 percent ). The MCF-7 cell number on the different biomaterial surfaces showed no difference when MC3T3 cells were not pre-seeded, indicating that the presence of MC3T3 cell is potentially beneficial for MCF-7 cell adhesion.

A 3D bone niche structure was printed from an extruded polymer-ceramic composite filament. The initial structure shows promise as an in vitro model of combined bone tissue and tumor for in-depth BCBM evaluations, e.g. early detection, intervention prevention, and treatment. Investigations of the modified MC3T3-conditioned environment, 3D cell culture in a printed scaffold with surface treatment, and controlled release of therapeutic drugs are ongoing to clarify the interactions of osteoblasts and breast cancer cells.

References:
1. Chen, YC, et al., Breast Cancer Res. 12, (2010).
2. Chen, WT, et al., BMES Annual Meeting (2023)

Acknowledgements (Optional): Funding for this work was provided by the Harbor Lights Endowment