(L-441) Micro-Computed Tomography for the Microstructure Evaluation of 3D Bioprinted Scaffolds

Thursday, October 12, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row L - Poster # 441

Presenting Author(s)

HB

Hannia V. Balcorta (she/her/hers)

Undergraduate Research Assistant
University of Texas at El Paso
El Paso, Texas, United States

Primary Investigator(s)

Wilson Poon

Assistant Professor
University of Texas at El Paso, United States

Last Author(s)

BJ

Binata Joddar

Associate Professor
University of Texas at El Paso, United States

Co-Author(s)

IH

Ivana Hernandez

Graduate Research Assistant
University of Texas at El Paso, United States
SR

Salma Ramirez

Undergraduate Research Assistant
University of Texas at El Paso, United States
PK

Piyush Kumar

Postdoctoral Fellow
University of Texas at El Paso, United States
VK

Vinod Kumar

Associate Professor
University of Texas at El Paso, United States

Introduction::

Tissue engineering aims to create 3D tissues in vitro that replicate native tissues in vivo for regenerative medicine (1). 3D bioprinting is a technique that can potentially enable the production of artificial cellular constructs that better mimic the complexity of human tissue, but accurately characterizing the microstructure of 3D bioprinted scaffolds remains challenging (2). Our objective in this study was to implement micro-computed tomography (micro-CT) with gold nanoparticles (AuNPs) to explore the microarchitecture and mechanical properties of 3D alginate-gelatin hydrogel scaffolds with cells. The complex microstructure and biomechanics of tissues is important in supporting the biological and structural functions of the organ (3). By understanding the microstructure of 3D bioprinted tissue engineering scaffolds using micro-CT and AuNPs, we can devise methods to optimize the 3D bioprinting process to improve cell distribution and proliferation within scaffolds. In this manner, we would be able to produce more physiologically and functionally relevant artificial constructs for regenerative medicine applications.

Materials and Methods::

To address the objective, we used polyethylene glycol-coated 60 nm AuNPs, sodium alginate, and gelatin to constitute the bio-ink with AC16 cardiomyocytes. 3D scaffolds were bioprinted using CELLINK BIO X with varying AuNP concentrations to optimize for micro-CT contrast and cell viability (4, 5). We used UV-Vis spectrophotometry, dynamic light scattering (DLS), and transmission electron microscopy (TEM) to characterize AuNPs and their concentration. To assess if the AuNPs affected cell viability, we performed live-dead assay on 2D cell cultures and 3D bioprinted cell-laden scaffolds with AC16 cardiomyocytes. We used scanning electron microscopy (SEM) and micro-CT to visualize scaffold microstructures with varying concentrations of AuNPs to quantify the imaging contrast improvement; as well as with and without AC16 cardiomyocytes to investigate microstructure changes from cellular remodeling.. ImageJ and DragonFly ORS Software were used to generate 3D reconstructions. Mechanical properties of the 3D bioprinted scaffolds were measured using rheometry and compared to values derived from micro-CT reconstruction.

Results, Conclusions, and Discussions::

The implementation of AuNPs as contrast agents significantly improved micro-CT image resolution in 3D bioprinted scaffolds. Cell viability of AC16 cardiomyocytes was unaffected by AuNPs in 2D and 3D cell culture conditions at all concentrations tested. However, high AuNP concentration (0.22 OD) was required for optimal micro-CT contrast in the scaffolds, leading to visible micro-aggregates in SEM images. Incorporating AuNPs and AC16 cardiomyocytes reduced storage/loss modulus, complex viscosity, and elastic modulus of the 3D bioprinted scaffolds by 2-3 times. Micro-CT images revealed scaffold biodegradation during in vitro incubation with cells and changes in mechanical parameters.

The positive outcomes highlight the importance of using AuNPs in micro-CT imaging for 3D bioprinted scaffolds. Enhanced resolution aids scaffold microstructure visualization, optimizing tissue regeneration and cell dispersion in future experiments. The absence of cytotoxicity in AC16 cardiomyocytes ensures the safety of AuNPs as contrast agents. However, evaluating cellular scaffold microstructure revealed deterioration over time, affecting scaffold stability during in vitro culture and necessitating further optimization to support proper physiological function once implanted in vivo for patients.

In conclusion, micro-CT with AuNPs as contrast agents significantly advances the characterization of 3D bio-printed scaffold microstructure. This improved imaging technique is a valuable tool for optimizing tissue-engineered scaffolds, potentially leading to more successful tissue regeneration and repair. The biocompatibility of AuNPs with cardiomyocytes enhances their safety for tissue engineering applications. Addressing scaffold stability concerns requires additional research.

The impact of this study extends to tissue engineering and regenerative medicine. The non-destructive, high-resolution micro-CT using AuNPs allows accurate determination of scaffold microstructure and mechanical properties. This has the potential to advance 3D scaffold design and manufacturing for tissue repair and regeneration. Understanding scaffold microstructure in 3D bioprinting can wide applications for treating a variety of diseases and injuries since each organ or tissue requires a specific microstructure and cell patterning for proper physiological function.. Ultimately, this research may contribute to patient-specific tissue engineering solutions for personalized approaches in regenerative medicine.

Acknowledgements (Optional): : The authors would like to acknowledge Alex Frickenstein and Dr. Stefan Wilhelm at the University of Oklahoma for performing TEM for our study. We also would like to thank Zayra N. Dorado for help with SEM. We thank Brittany Payan Baca and Dr. Natividad-Diaz for technical assistance in rheological analysis. For the Micro-CT analysis, we are grateful to Dr. Jessie Maisano at UTCT (Austin). This study was supported in part by the National Institutes of Health, NIH 1SC1HL154511-01 and the National Science Foundation, NSF 1927628 grants to BJ. SPR acknowledges the COURI MERITUS UG research fellowship at UTEP from fall 2022-spring 2023 for support. WP and HB acknowledge support from the University of Texas Systems STARs Program and the University Research Institute (URI) award 14648682 from the University of Texas at El Paso.

References (Optional): :

1. Khademhosseini, A. and R. Langer, A decade of progress in tissue engineering. Nature protocols, 2016. 11(10): p. 1775-1781.

2. Ikada, Y., Challenges in tissue engineering. Journal of the Royal Society Interface, 2006. 3(10): p. 589-601.

3. Chen, P.-Y., et al., Structure and mechanical properties of selected biological materials. Journal of the mechanical behavior of biomedical materials, 2008. 1(3): p. 208-226.

4. Wu, Y., et al., Gold nanoparticles in biological optical imaging. Nano Today, 2019. 24: p. 120-140.

5. Joddar, B., et al., Delivery of mesenchymal stem cells from gelatin–alginate hydrogels to stomach lumen for treatment of gastroparesis. Bioengineering, 2018. 5(1): p. 12.