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Tissue Engineering

Tissue Engineering - Poster Session E

Z14 - Evolving Scaffold Design for Physiological Relevance in a 3D Printed Bone Niche Model

Saturday, October 11, 2025
10:00 AM - 11:00 AM PDT
Location: Exhibit Hall F, G & H

Introduction: : The skeletal system is the most common site of breast cancer metastasis, with bone metastases occurring in approximately 70% of patients with advanced disease stages [1]. The porous architecture of bone, particularly when altered by osteolytic lesions, is hypothesized to facilitate tumor cell colonization, adhesion, and proliferation. Understanding the relationship between bone porosity and cancer cell behavior is essential for the development of physiologically relevant in vitro models of the metastatic bone microenvironment and for the advancement of targeted therapeutic strategies. Benchmarking is necessary to evolve scaffold design and determine physiological relevance. In this study, polyethylene terephthalate glycol (PETG) and nano-hydroxyapatite (nHA) were used to fabricate a porous, composite scaffold to mimic the chemical component and geometrical properties of native cancellous bone. It was hypothesized that scaffolds fabricated from PETG-nHA with higher porosity (lower infill density), that better mimic the osteoporotic condition, would support higher levels of bone cell adhesion and growth. It was further hypothesized that scaffolds with nHA would curb growth of MCF-7s as a benchmark of the composite material [2].

Materials and

Methods: : A composite material (denoted as PETG-nHA) of 70wt% PETG and 30wt% nHA was extruded to obtain a filament with a diameter of 1.65-1.85 mm. As-received PETG filament and the PETG-nHA filament were used in a PRUSA MK4 3D Printer to create porous scaffolds. Scaffolds were printed to approximate the porous densities of cancellous bone in different health conditions: 40% for healthy bone and 60% and 70% representing degenerated conditions. Six groups of scaffolds were printed, n=3 for each infill density group. The PETG scaffolds served as a negative control for testing the composite filament. The porosity profiles were evaluated with optical microscopy and ImageJ. Subsequently, 2×105 MCF-7 breast cancer cells were seeded on each scaffold in 75µL aliquots. Initial cell adhesion was inferred by alamarBlueTM assay, quantified as metabolism percent reduction. The scaffolds were incubated under standard conditions for five days. Differences in all the quantifiable results were assessed by t-test and one-way ANOVA with pairwise comparison.

Results, Conclusions, and Discussions:: The average pore sizes of the scaffolds were as follows: 70%: 63 ± 10µm, 60%: 107 ± 18 µm, 40%: 347 ± 27 µm, corresponding to the designed sizes of 70%: 69µm, 60%: 118µm, 40%: 361µm (Fig. 1). The Alamar Blue assay results on Day 5 were inconclusive because the cellular activity exceeded the stain saturation limit. On Day 1, cellular PETG scaffolds resulted in higher fluorescence values across all infill densities, indicating the higher initial cell metabolic activity, suggesting higher adhesion (Fig 2). The composite scaffolds displayed significantly lower initial cell metabolic activity for all infill densities (p < 0.05) (e.g. PETG_40% vs composite_40%: 77 ± 10% vs 96 ± 11%), suggesting corroboration with earlier studies that showed inhibition of MCF-7 proliferation on nHA particles2. Although initial cell metabolic activity was generally high (from 73 ± 6% for PETG-nHA-70% to 99 ± 3% for PETG-70%) across all the groups, the cell metabolic activity was not significantly higher on Day 3 than on Day 1 for all the groups. It is mildly possible the relatively high passage number (19) of MCF-7 cells used in this study might result in a lower metabolic activity. Moreover, the initial cell metabolic activities on different infill densities with the same material were not significantly different (e.g. composite-60% vs composite-40%: 76.5 ± 4.7% vs 76.9 ± 10.3%), suggesting that the variations in infill densities did not affect MCF-7 cell metabolic activity in this study.

The success in 3D printing a range of pore sizes suggests that this process can be applied to a variety of applications in the future. To better understand potential differences in cell adhesion and proliferation on the two types of scaffolds, cell number must be calculated. As there were no statistical differences in cell metabolic activity for different infill densities and days, future studies must be conducted, for example, increasing the intervals of infill densities, prolonging cell culture time, using MCF-7 cells with lower passage number, to further assess if pore size affects MCF-7 adhesion and/or growth and if, indeed, the 3D bone niche model demonstrates physiological relevance.

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

References:
1. Sanmartin, M, et al. Bone marrow/bone pre-metastatic niche for breast cancer cells colonization: The role of mesenchymal stromal cells. Critical Reviews in Oncology/Hematology, 164 (2021)
2. Fukada, M, et al. Size and morphology-mediated antiproliferative activity of hydroxyapatite nanoparticles in human breast cancer cells. Journal of Nanotechnology 2023:1 (2023)