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High School Student with a Traditionally Underrepresented Background

HS Poster

Poster 23 - Application of 3D-Bioprinting for the Development and Characterization of Hydrogels for Chondrocyte Regeneration

Saturday, October 26, 2024
10:00 AM – 12:00 PM EST
Location: Exhibit Hall E, F & G

    Presenting Author(s)

  • Ankur Rao

    Senior @ Green Level High School & Researcher
    Clemson University
    Apex, North Carolina, United States

    • Co-Author(s)

  • Larry Y. Liu (he/him/his)

    Senior @ Green Level High School and Researcher
    Clemson University
    Cary, North Carolina, United States

    • Co-Author(s)

  • Wesley Nichols

    Ph.D. Bioengineering Student and Researcher
    Clemson University, United States

  • Vishal J. Thomas, PhD

    Lecturer
    Clemson University
    Central, South Carolina, United States

  • Lucas Schmidt, PhD

    Lecturer and Researcher in Optical Imaging
    Clemson University, United States

    • Primary Investigator(s)

  • Bruce Gao, PhD

    Professor and The South Carolina SmartState Endowed Chair in Biofabrication Engineering
    Clemson University, United States

    • Presenting Author(s)

  • Larry Y. Liu (he/him/his)

    Senior @ Green Level High School and Researcher
    Clemson University
    Cary, North Carolina, United States


Introduction: The field of regenerative medicine has made significant strides in developing solutions for cartilage repair. Hydrogels have emerged as promising materials in this field because they mimic the extracellular matrix (ECM) and deliver bioactive molecules to cells in various tissue engineering applications. Simultaneously, bioprinting advances in cell placement and structural formation have made new medical device concepts possible. Despite the concept’s potential, the clinical adoption of bioprinted hydrogel-based therapies is often hampered by challenges of the hydrogel, such as suboptimal mechanical strength and poor tissue integration. Recent studies have explored the use of hydrogels in chondrocyte cell growth but have not considered their mechanical stiffness in regards to bioprinting applications. In this study, we aim to develop crosslinked hydrogel scaffolds through bioprinting to enhance structural integrity and functional performance, bridging the gap between the lab and clinic. We aim to assess hydrogel’s printability through a bioprinter, its ability to support chondrocyte regeneration through advanced analytical methods, including Atomic Force Microscopy (AFM), and its morphological effect on chondrocyte cells. A more comprehensive understanding of hydrogel performance opens up new possibilities for developing innovative medical devices to enhance tissue regeneration and repair, addressing shortcomings in current treatments.

Materials and

Methods: Various hydrogel formulations were synthesized by incorporating biopolymers such as alginic acid sodium salt, agarose, porcine skin, and gelatin. These formulations were subjected to chemical crosslinking using EDC-NHS coupling, glutaraldehyde fixation, and riboflavin photoinitiation to enhance their mechanical properties. Hydrogel candidates with optimal characteristics for chondrocyte proliferation were identified based on preliminary in-vitro assays. The gelation kinetics were monitored over a 24-hour period. Hydrogel samples were subjected to Atomic Force Microscopy (AFM) to evaluate their mechanical properties. AFM is a high-resolution imaging technique that utilizes a sharp probe tip mounted on a cantilever to scan a sample surface. The cantilever with a sharp probe tip was oscillated near its resonance frequency, causing intermittent contact with the hydrogel surface. The AFM system "tapped" 100 discrete locations across the hydrogel samples to generate detailed force maps. The collected data were processed using MATLAB to compute the Young's modulus of the hydrogels, which quantified the material's tensile and compressive stiffness. To mimic cartilage tissue, we aimed to convert these gels into scaffolds using additive manufacturing methods. Promising hydrogels were extruded through a dual-nozzle bioprinter which we adapted to handle the unique properties of our hydrogel by optimizing extrusion parameters such as speeds, offsets, and pressures. Hydrogel compositions that met our desired criteria were placed in petri dishes filled with low-glucose Dulbecco's Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) and 1% AA, and seeded with porcine chondrocyte cells.

Results, Conclusions, and Discussions: The novel hydrogel composition candidate, consisting of 20% (w/v) porcine skin gelatin and 2% (w/v) agarose, with 1% glutaraldehyde as the crosslinking agent, effectively transitions between mechanical property stages. AFM analysis of the printed scaffolds indicated enhanced structural integrity, with a Young’s modulus value comparable to that of healthy human cartilage (0.05 ±.005 MPa), as seen in Figure 1 , which is promising for applications where mechanical robustness is critical. This achieves the necessary stiffness for chondrocyte support while retaining the extrudability required for precise 3D bioprinting. Initial seeding of the hydrogels (85% - 90% confluency) with chondrocytes has shown promising signs of cell viability, growth support, and morphological changes that are indicative of healthy proliferation, as seen in Figure 2. These findings highlight the potential of our bioprinted hydrogels to serve as a foundation for future clinical applications, particularly in the development of accessible, implantable solutions for cartilage repair. Ongoing work will refine the bioprinting parameters and further explore the clinical applicability of this technology, with the goal of creating a fully functional, bioprinted implant for in-vivo use.

Acknowledgements (Optional): This research is supported by Clemson University and the National Science Foundation (OIA-2242812).

References (Optional): Smith, J. A., et al. (2020). Enhancing mechanical properties of hydrogels for tissue engineering applications: A crosslinking approach. Biomaterials Science, 8(2), 432-445.

Cheng, N. C., Estes, B. T., Young, T. H., & Guilak, F. (2021). Engineered cartilage with biological and mechanical properties mimicking native tissue: A milestone in the evolution of cartilage repair strategies. Regenerative Biomaterials, 8(4), rbab030. https://doi.org/10.1093/rb/rbab030

Feng, X., McDonald, J. M. (2017). Disorders of bone remodeling. Bone Research, 5, 17017. https://doi.org/10.1038/boneres.2017.14

Yang, L., Tsang, S. H., & Wang, S. (2019). Micro/nano-fabrication technologies for retinal tissue regeneration. Regenerative Biomaterials, 6(3), 129-139. https://doi.org/10.1093/rb/rbz013

Chen, Q., Liang, S., & Thouas, G. A. (2017). Elastomeric biomaterials for tissue engineering. Polymers, 9(12), 671. https://doi.org/10.3390/polym9120671