I12 - Fabrication and Characterization of Collagen-coated Polyethylene Terephthalate Glycol-nano-Hydroxyapatite (PETG-nHA) Scaffolds for Reproducible Breast Cancer Bone Metastasis Models

Introduction: : Bone is the most frequent site of metastasis, involved in approximately 70% of all breast cancer metastatic cases. [1] Breast cancer bone metastasis (BCBM) typically initiates within the bone marrow, a microenvironment rich in mesenchymal stem cells (MSCs) [2] housed in the porous cavity of cancellous bone. Native cancellous bone is characterized by its complex porous and trabecular architecture, comprising an inorganic mineral phase and an organic matrix, primarily composed of type-I collagen (Col). Modeling this intricate structure with its essential biochemical components in vitro remains a significant challenge. In this study, we developed a 3D printed scaffold designed to model key structural and chemical features of native bone. The study objective is to demonstrate the potential of a scaffold to form an MSC-favorable cancellous bone environment in vitro, providing a platform to study early-stage interactions between breast cancer cells and MSCs during BCBM progression. Scaffolds were developed using polyethylene terephthalate glycol (PETG) polyester incorporated with nano-hydroxyapatite (nHA) particles to replicate the inorganic mineral phase. To improve the scaffold surface hydrophilicity and mimic the organic microenvironment, scaffold surfaces were coated with Col to favor biological activities such as cell attachment and proliferation and interactions with bone-resident cells.

Materials and

Methods: : A composite of 70wt% PETG and 30wt% nHA was prepared [3] and then extruded into a 1.75mm diameter filament at 195°C. The filament was used in a standard 3D printer to generate cylindric trabecular scaffolds with a gyroid pattern mimicking normal cancellous bone geometries i.e., with 60% infill density and open pores (7×5 mm diameter × height ). Three different groups of scaffolds were obtained. The first group was fabricated from PETG alone (i.e., “PETG”) and the second from PETG and nHA (i.e., “PETG-nHA”). The third group was generated by printing PETG-nHA scaffolds, immersing them (24hrs) in a dopamine solution (pDA, 1mg/ml) and coating them with Col (2.5% w/v, 1hr) (i.e., “PETG-nHA/Col”). Scaffold hydrophilicity was measured as water contact angle (WCA). Subsequently, scaffolds placed in 24-well plates were seeded with MSCs (1×105 cells in 75µL of culture medium), cultured for 30 min., and washed with fresh medium to remove the unbound cells. Wells with no scaffolds and seeded with MSCs (1×105) served as positive controls. The percent MSC cell adhesion was estimated (indirectly) using Alamar Blue staining. Both hydrophilicity and cell adhesion were compared in repeated independent experiments (n ≥4) using a one-way ANOVA and post-hoc test with a p>0.05 threshold.

Results, Conclusions, and Discussions:: The PETG-nHA composite material was successfully extruded into filaments for use in 3D printing and printed into scaffolds. The porosities (57.9 ± 0.15% vs. 60%) and the width of struts of all the as-printed scaffolds were similar to the as-designed porosity and width of struts, respectively (Fig 1), thus demonstrating increased printing fidelity compared to prior attempts at fused deposition modeling. [4] The hydrophilicity, as measured by WCA, markedly increased following nHA coating of PETG scaffolds and further increased when scaffolds were further coated with CoI (136.3 ± 1° vs. 79.5 ± 0.9° vs. 0°, Fig. 2), respectively, indicating the effectiveness of nHA and Col coating in significantly improving hydrophilicity. [5] As expected, MSC cell adhesion to PETG scaffolds was low compared to MSC cell adhesion to PETG-nHA and PETG-nHA/Col scaffolds (3.57 ± 0.63% vs. 4.41 ± 0.42% vs. 4.88 ± 0.45%, Fig 3, p< 0.05), suggesting that the presence of both nHA and CoI improves cell adhesion. [6] Interestingly, PETG-nHA/Col scaffolds led to MSC adhesion equivalent to 75% of the cell adhesion observed in tissue culture treated well plates (i.e. 4.88 ± 0.45% vs. 6.35 ± 0.41%, p< 0.05) suggesting the benefit of scaffold nHA and CoI treatments in promoting cellular adhesion. [7]

Acknowledgements and/or References (Optional):: Acknowledgments: Funding for this work was provided by the Harbor Lights Endowment.
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