Poster H2 - Assessing Cellular Calcium Deposition in 3D Printed Bone Models

Thursday, October 24, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Kosi Okeke (she/her/hers)

Undergraduate Researcher
University of Georgia
Athens, Georgia, United States

Co-Author(s)

CC

Caitlin Conyers

Undergraduate Researcher
University of Georgia, United States
Weitong Chen, Doctor of Philosophy (he/him/his)

Research Associate
Department of Small Animal Medicine & Surgery, University of Georgia
Athens , Georgia, United States
Timothy Burg

Professor
Department of Biomedical Sciences, University of Georgia
Athens, Georgia, United States

Last Author(s)

Karen Burg

Harbor Lights Endowed Chair
University of Georgia
Athens, Georgia, United States

Introduction: Bone metastasis is a common and complex aspect of breast cancer, affecting nearly 70% of patients. Engineered tissue could provide valuable insights into breast cancer bone metastasis and help develop effective treatments [1]. This study focuses on creating tissue-engineered scaffolds as the basis for in vitro models that recapitulate salient aspects of human bone to facilitate deeper understanding of breast cancer metastasis. [2]. It is hypothesized that cells seeded on 3D printed samples made with polyethylene terephthalate glycol (PETG) and β-tricalcium phosphate (β-TCP) will result in higher calcium deposits compared to scaffolds made solely with PETG.

Materials and

Methods: The samples were printed using a substrate made of pure PETG and a second layer structure made of either PETG or a polymer-ceramic mixture of PETG and β-TCP. This mixture was created by dissolving PETG in dichloromethane, adding β-TCP, generating a homogenous solution, drying at 45°C, and extruding the novel material to create filament for 3D printing. Three structures were printed: a raised letter “U” made of PETG on the PETG substrate, a raised letter “G” made of PETG+TCP on the PETG substrate, and a raised letter “A” made of PETG on the PETG substrate. The printed materials were then seeded with human mesenchymal stem cells, which were subsequently tested with alamarBlueTM and Alizarin Red assays. For the alamarBlueTM assay, 30µl of alamarBlueTM reagent was directly added to the cells in 270µl culture medium, incubated for 1-4 hours at 37°C away from light, and the absorbance was measured at 570nm, with 600nm reference wavelength. For Alizarin Red staining, the spent medium was removed, followed by a 10-minute treatment with 20 ml of methanol cooled at -20°C. The cells were then fixed using 500μL of methanol at -20°C for 5 minutes, washed three times with phosphate buffered saline, and stained with 300μL of Alizarin Red for 10 minutes before observing the scaffolds.

Results, Conclusions, and Discussions: The alamarBlueTM assay revealed a pink color only for the “G” sample, indicating high cell proliferation and viability, likely due to the presence of β-TCP. This result was confirmed by the highest cell count for the “G” sample (red bar in Figure 1), while “U” (black bar in Figure 1) and A (white bar in Figure 1) samples had no cell counts. After staining with Alizarin Red, the “G” scaffolds turned red, while “U” and “A” scaffolds remained white. This color change confirms the presence of calcium in the ceramic scaffolds. The ceramic makeup of the “G” scaffolds promotes calcium deposition, which Alizarin Red binds to, resulting in the red coloration of the “G” sample.

In conclusion, our study demonstrates that engineered scaffolds made with PETG, and β-TCP enhance cell growth and calcium deposition, as confirmed by alamarBlueTM and Alizarin Red staining, thereby, closely mimicking one select aspect of the natural environment of human bone. These results show the capability of engineered scaffolds to imitate interactions within the bone, providing insights into breast cancer metastasis to bone. By understanding these processes better, we can develop more effective treatments for breast cancer patients experiencing bone metastasis, improving their outcomes and quality of life.

Future Studies: In future studies advanced structures will be made using the composite material, incorporating pore size and shape variables in the 3D porous scaffold as the next step toward creating an in vitro bone model that can be used for further research addressing breast cancer and bone metastasis.

Acknowledgements (Optional): This work was supported by National Science Foundation Research Experiences for Undergraduates Grant #1950581. Additional funding was provided by the Harbor Lights Endowment.

References:
[1] Jackson W. III, Krishnan V., Mastro A.M. “The Breast Cancer Metastases in Bone Conundrum.” National Library of Medicine, 1 Jan. 2013, www.ncbi.nlm.nih.gov/books/NBK169226/.
[2] Ritchey B., Tissue Engineered Bone Models: Advances in 3D Printed Tricalcium Phosphate/Polylactide Scaffold, Thesis, 7 May 2024.