N23 - 3D Bioprinted Antibacterial and Cell Proliferative Hydrogel for Biomedical Applications

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

ER

Ethan Renken

Undergraduate Researcher
University of South Carolina, United States

Co-Author(s)

Aasma Sapkota

Graduate Research Assistant
University of Georgia
Athens, Georgia, United States

Primary Investigator(s)

Elizabeth J. Brisbois

Associate Professor
University of Georgia
Athens, Georgia, United States

Introduction: : Surgical site infections (SSIs) are the most common healthcare-associated infections, affecting approximately 20% of postoperative patients, leading to extended hospitalization, increased antibiotic usage, and heightened mortality risk[1]. SSIs primarily result from bacterial contamination due to insufficient or improper sterilization practices; however, current SSI dressings largely lack bioactive properties necessary to both prevent infection and actively promote tissue regeneration[2,3]. Additionally, the escalating challenge of antimicrobial resistance (AMR) further limits effective antibiotic therapies and emphasizes the need for alternative, multifunctional biomaterials in medicine and dentistry.
Nitric oxide (NO) is a bioactive gaseous molecule with potent antimicrobial efficacy, uniquely without risk for AMR development[4]. NO-releasing biomaterials can be fabricated through the incorporation of an NO donor molecule, which generates controlled amounts of NO gas in response to environmental stimuli, such as moisture or light. Additionally, an undisclosed active component (AC) with established cell-proliferative properties was incorporated into the novel material. With the inclusion of AC and a NO donor, this material synergistically combines antimicrobial potential and cell-proliferative capacity, making it well-suited for localized tissue repair after invasive surgical procedures.
Three-dimensional (3D) bioprinting is an additive manufacturing technique that utilizes cells, biomaterials, and biological molecules to create functional, bioactive constructs for regenerative medicine, implants, or disease modeling. Bioprinting offers several benefits over traditional manufacturing techniques, including cell-embedment to assist with tissue integration and patient specificity[5]. This platform facilitates the incorporation of a NO donor and AC within cell-laden hydrogels, yielding a multifunctional dressing that addresses SSIs and accelerates wound healing without risking AMR.

Materials and

Methods: : The primary hydrogel components include a biocompatible polymer backbone, a crosslinking agent, and an NO donor molecule, the composite formulation herein referred to as “Gel”. Additionally, AC was dispersed into the Gel solution (10 mg/mL) (Gel-AC) and both sample types were crosslinked under UV light (405 nm) for 5 minutes. Sample groups Gel and Gel-AC were analyzed for their NO releasing ability with a Nitric Oxide Analyzer (NOA) (NOA 280i, Zysene) (data not shown). Bactericidal capacity was measured by incubating samples with Staphylococcus aureus (S. aureus) for 24 h at 37℃ and counting colonies to quantify the number of planktonic bacteria (data not shown).
To assess the bioactivity of AC, a transwell system was used to expose human cells to the following concentrations of AC in phosphate buffered saline (PBS): 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.0625 mg/mL, and 0 mg/mL. Cell proliferation was measured using an MTT assay at the 2-, 4-, and 7-day timepoints.
For cell-laden printed gel fabrication, cells were added to the precursor Gel and Gel-AC solutions for extrusion-based printing and UV crosslinking using 3D bioprinter (Bio X, Cellink). Cell-laden gels were incubated at 37℃ and 5% CO2 for 2, 4, and 7 days. Cell proliferation was quantified using an alamarBlue metabolic assay (data not shown).

Results, Conclusions, and Discussions:: NO release measurements found Gel-NO-AC samples exhibited greater NO flux than Gel-NO (data not shown). Additionally, these flux levels are comparable to physiologically relevant values ranging from 0.5 to 4.0 x 10-10 mol · cm-2 · min-1, indicating therapeutic potential. We hypothesize the addition of AC increases the porosity of the material matrix and swelling capacity, potentially leading to higher NO flux.
After a 24 h incubation with S. aureus, both NO-releasing samples (Gel-NO and Gel-NO-AC) significantly reduced the amount of planktonic bacteria load (data not shown). These results indicate that the addition of AC to the NO-releasing hydrogel platform does not interfere with the well-characterized antibacterial nature of a nitric oxide-releasing biomaterial.
Active component indirect contact assays concluded AC promoted significant, dose-dependent cell proliferation at 2-, 4-, and 7-day timepoints, with higher concentrations amplifying the effect (Figure 1). Furthermore, cell-laden, AC-doped, printed hydrogels demonstrated a similar trend with significantly increased cell counts at all three timepoints compared to AC-free samples (data not shown). Both results strongly convey the proliferative effects experienced by human cells when exposed to AC.

In this study, a NO-releasing hydrogel containing a cell-proliferative active component (AC) was fabricated, achieving physiological levels of NO release in the first 24 h. These gels demonstrated excellent antibacterial activity with significant reduction in planktonic S. aureus viability. Additionally, AC was confirmed to promote cell proliferation both when exposed to cells independently and when incorporated into a 3D bioprinted, cell-laden hydrogel. These results support the material’s dual bioactive role and therapeutic potential.
In future studies, we plan to investigate AC influence on cell activity through the measure of key protein markers. Furthermore, we hope to examine the immunomodulatory functionality of AC, as well as the potential osteoimmunomodulatory signaling between immune cells and cells in damaged tissue.

Acknowledgements and/or References (Optional): : 1. Centers for Disease Control and Prevention. (2023). Surgical Site Infection (SSI) Event. National Healthcare Safety Network (NHSN) Patient Safety Component Manual.
2. Rezaei AR, Zienkiewicz D, Rezaei AR. Surgical site infections: a comprehensive review. J Trauma Inj. 2025 Jun;38(2):71-81. doi: 10.20408/jti.2025.0019. Epub 2025 Jun 27. PMID: 40571954; PMCID: PMC12229807.
3. Liu, Y., et al. (2024). Multifunctional biomimetic hydrogel dressing provides anti-infection and tissue regeneration for wound healing. International Journal of Pharmaceutics, 642, 123123 DOI: 10.1111/wrr.13233
4. Okda M, Spina S, Safaee Fakhr B, Carroll RW. The antimicrobial effects of nitric oxide: A narrative review. Nitric Oxide. 2025 Apr;155:20-40. doi: 10.1016/j.niox.2025.01.001. Epub 2025 Jan 8. PMID: 39793728.
5. Mirsky NA, Ehlen QT, Greenfield JA, Antonietti M, Slavin BV, Nayak VV, Pelaez D, Tse DT, Witek L, Daunert S, Coelho PG. Three-Dimensional Bioprinting: A Comprehensive Review for Applications in Tissue Engineering and Regenerative Medicine. Bioengineering (Basel). 2024 Jul 31;11(8):777. doi: 10.3390/bioengineering11080777. PMID: 39199735; PMCID: PMC11351251.