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Tissue Engineering

Tissue Engineering - Poster Session D

Poster K18 - Optimization and Validation of 3D Nanoscale Tissue Engineered Bone Model

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Ewing Sarcoma (ES) is a pediatric bone and soft tissue cancer with a survival rate below 20% for metastatic cases (Fong, 2022). This is due to ineffective treatments, as promising therapeutics often fail clinical trials because of inadequate research models. Current research relies heavily on 2D cell cultures, which fail to replicate the complex 3D bone tissue environment, hindering therapy development.

Mechanical properties of tissue microenvironments are crucial in ES pathology, which is not recapitulated in 2D culture. ES cells express high levels of matrix metalloproteinases (MMPs), which break down collagen and other extracellular matrix components (Paiva, 2017). ES cells create pathways for migration, increasing localized stiffness and weakening the structure (Prockop, 1995) and secrete factors that stimulate surrounding cells to modify the matrix (Scotlandi, 1998). A 3D culture is necessary to study these behaviors; however, ES researchers rarely use them, leading to an approach gap.

To address these challenges, we developed an emulsion-based 3D microscale bone model using collagen and hydroxyapatite (HA) to encapsulate cells in a spherical matrix model. This model aims to mimic the physiological environment more accurately. Optimization involved testing different compositions: pure collagen, collagen with calcium ions, and collagen with HA. Validation included micropipette aspiration to quantify model stiffness with and without ES cells, observing cell-initiated modifications within the model. We hypothesize models with longer-cultured ES cells will be less stiff. The model offers a relevant platform for studying tumor behavior and testing treatments, enhancing ES research, accelerating therapy development, and ultimately improving patient outcomes.

Materials and

Methods:  Collagen (8.2 mg/mL rat tail, Corning) was kept on ice and 0.5 mg/mL calcium nitrate tetrahydrate ions or HA were added and afterwards neutralized with NaOH to a pH of 7.4±0.05. Cells (GFP expressing TC-71, courtesy of Yustein lab, Emory University) were cultured to 80%-90% confluence, detached with trypsin, and resuspended to a density of 1 million cells per mL of collagen.

The cell-collagen mixture is added dropwise to the preheated oil mixture containing 0.5% Span80 v/v, then agitated at a speed of 40 RPM orbital agitation to create an emulsion. Pure collagen spheres required 45 minutes of agitation, the HA prototype needed 20 minutes, and the ion prototype took 45 minutes. The spheres were then filtered using a 40 µm cell strainer and imaged using a Nikon Eclipse Ti2 inverted microscope at 4x magnification. Images were analyzed with a custom Matlab script to determine sphere diameter, circularity index, and cell encapsulation efficiency.

Glass micropipettes were made using thin walled pipettes which were pulled using a P-97 Pipette Puller. The pipettes had tip diameters of 20 µm which were created via a microforge. The spheres are mounted on an aspiration chamber set-up and pressure was applied via suction. Images and pressure measurements were taken to calculate the effective Young’s Modulus of the models.

Results, Conclusions, and Discussions: The mean sphere diameters for collagen-only, ion, and HA prototypes were 387.5 µm, 280.4 µm, and 246.4 µm, with standard deviations of 325.4 µm, 207.9 µm, and 155.3 µm (Figure 1). A Tukey-Hockey Post Hoc test showed significant differences between collagen-only and HA prototypes (p < 0.0001) and between collagen-only and ion prototypes (p < 0.0001).

Circularity indexes, indicating how close the spheres are to a perfect circle (score of 1), were 0.24 for collagen-only, 0.64 for HA, and 0.73 for ion prototypes. Significant differences were found between all prototypes (p < 0.0001), with the ion prototype being closest to a perfect circle, suggesting high replicability and stability. The HA prototype produced the smallest and most uniform spheres, making it optimal for future work involving single-cell devices.

Stress-strain curves generated via ImageJ and pressure measurements for the effective Young’s Modulus of spheres made without cells (n=6) is 8.47x10^2 Pa with a standard deviation of 6.47x10^2 Pa, and with cells (n=7) is 4.99x10^2 Pa with a standard deviation of 1.24x10^2 Pa (Figure 2). There was no significant difference (p < 0.1889) between spheres with and without cells.

The HA prototype showed superior sphere uniformity and circularity, making it the best candidate for further research. HA's addition produces smaller, more circular spheres by depositing crystals between collagen fibrils, promoting interlocking and stability. Future work involves mechanical testing of HA prototypes with ES cells cultured over several days using micropipette aspiration to assess sphere remodeling. Preliminary results show no significant differences between spheres made with and without cells, however the standard deviation of spheres without cells was much larger than spheres with cells. The high variation in modulus values in control is attributed to local differences in HA composition.

The model aims to provide a more physiologically relevant platform for studying tumor behavior and testing treatments, with the HA prototype's performance highlighting the importance of biomimetic materials. Refining and validating this model will advance studies of mechanosensitive cell types and interface with various omics techniques like RNA sequencing.

Acknowledgements (Optional):  I would like to express my profound gratitude to my research mentor, Jocelyn Baker, B.S., for her extensive contributions to the optimization and validation of the protocol for our 3D nanoscale tissue-engineered bone model. I would also like to thank Dr. Robert Raphael, Ph.D., for his knowledge and expertise regarding micropipette aspiration. Additionally, I extend my thanks to my principal investigator, Dr. Julea Vlassakis, Ph.D., who conceptualized and guided the overarching project.

The ES cell lines crucial for this project were generously provided by the Texas Children’s Hospital Faris D. Verani Ewing Sarcoma Research Center, in a collaborative effort led by Dr. Jason Yustein, M.D., Ph.D., with significant contributions from Ryan Shuck and Atreyi Dasgupta, Ph.D. We also utilized research equipment and facilities at the Rice Nanofabrication Cleanroom and the Shared Equipment Authority, which were essential to our work.