Poster AA16 - Novel Three-Dimensional Printed Piezoelectric Breast Cancer Bone Metastasis Model

Introduction: Bone has been identified as the most prevalent site for breast cancer metastasis; however, the cause is not fully understood. In vivo, bone tissue experiences mechanical loading and creates electrical signaling as a result of piezoelectric properties. Evidence suggests that breast cancer cells undergo increased proliferation and migration in the presence of electrical fields caused by the electrical signaling of bone cells. The development of three-dimensional (3D) models have been widely investigated in an attempt to better understand the underlying mechanisms for breast cancer metastasis; however, current models lack a piezoelectric component. This study aims to develop a 3D in vitro piezoelectric breast cancer bone metastasis model to more accurately observe the interactions between breast cancer cells and the bone microenvironment. Our model consists of a 3D printed scaffold made of polycaprolactone (PCL), demineralized bone matrix (DBM), and polyvinylidene fluoride (PVDF). Preliminary results show that our PVDF-loaded scaffold provides a piezoelectric effect within the model when subjected to mechanical stimulation. A transwell migration assay was used to compare the migratory behavior of low-metastatic (MCF-7) and high-metastatic (MDA-MB-231) breast cancer cells. Recent findings indicate that the addition of a piezoelectric component into the model preserved metastatic behavior. This work indicates that the addition of a piezoelectric component increases the physiological similarity of in vitro modeling systems. With a more physiologically accurate modeling system, novel cancer treatments and breast cancer behavior can be investigated.

Materials and

Methods: To create the 3D piezoelectric model, 10% wt/vol solutions of PCL in dichloromethane and PVDF in dimethylformamide were created, then mixed together until homogeneity was reached. Bioactive composites were created via ambient wet mixing of the DBM powder and the polymer solution at an optimized weight ratio of 50:50. Then, scaffolds were fabricated by 3D pneumatic printing the composite solution at ~120 kPa. After printing, the structure was submerged in deionized water during the critical drying period to enhance piezoelectric properties. Printed films were dried, and after full solvent and water evaporation, films were cut into circular scaffolds (16-mm diameter) with a metal punch and press fit into 24-well plates. Scaffolds were pre-conditioned in media overnight.

A transwell migration assay was used to determine the efficacy of the model. Murine preosteoblast bone cells (MC3T3-E1) were seeded onto the fabricated scaffolds in the lower chamber of transwell inserts then, differentiated for 7 days to develop an extracellular matrix within the models. Models were subjected to mechanical stimulation via ultrasonic treatment to stimulate the piezoelectric component and promote osteogenic differentiation. Xylenol Orange staining and a Cell Counting Kit-8 (CCK-8) assay were performed at Day 3 and 7 to observe osteogenic differentiation. Breast cancer cells (MCF-7 and MDA-MB-231) were subsequently seeded onto the upper chambers. After cell adhesion, the treatment group was subjected to 30 minutes of ultrasonic stimulation. The number of migrated breast cancer cells was determined by cell count at 6- and 12-hour timepoints.

Results, Conclusions, and Discussions: Xylenol Orange staining showed an increase in calcium deposits from Day 3 to Day 7, confirming that bone cells had differentiated within the models. CCK-8 data showed no significant change from Day 3 to Day 7, providing additional evidence that the bone cells were differentiating instead of proliferating.

Breast cancer cell behavior (MCF-7 and MDA-MB-231) was compared by performing a cell count of the number of cells that migrated through the transwell membrane. At 6 hours after breast cancer cell attachment, a greater number of MDA-MB-231 cells had migrated compared to MCF-7 cells in the group receiving ultrasonic treatment. The control group, not receiving ultrasonic treatment and therefore not containing a piezoelectric component, did not display any significant difference in the number of migrated MDA-MB-231 and MCF-7 cells. At hour 12, ultrasonic treatment did not create a difference in migratory behavior. These results indicate that the migratory behavior of breast cancer cells was preserved within the model at early stages, only when piezoelectricity was present. Thus, the addition of a piezoelectric material could significantly improve the physiological effects observed in a 3D in vitro model.

This study successfully demonstrated the feasibility of a dynamic biomimetic 3D bone model. The results obtained in this study indicate that the addition of piezoelectric materials could significantly improve the accuracy of 3D in vitro models. Metastatic behavior was displayed in our dynamic model as exhibited by the increased number of cancer cells that migrated towards the bone niche. With an effective in vitro model, we can begin to investigate novel cancer treatments and gain a deeper understanding of the mechanisms of breast cancer metastasis to bone. Future work will include testing the effects of continuous ultrasound during a shorter period of time and optimizing the voltage output of the piezoelectric scaffold for enhanced physiological effects.

Acknowledgements (Optional): Many thanks Dr. Damion Dixon for technical assistance. Funding for this work provided by the National Science Foundation CAREER Award #2145521.