(A-4) Printing GelMA Bioinks: A Strategy for Building in vitro Model to Study Nanoparticle-based Minocycline Release and Cellular Protection under Oxidative Stress

Introduction:: Minocycline (MH) is a member of the tetracycline antibiotics with broad-spectrum antimicrobial activity. Additionally, it has demonstrated anti-inflammatory and anti-apoptotic properties, and has been widely applied in treating infections, inflammation, and neurodegenerative diseases. However, for topical wound dressings or drug-loaded implants, customization of shape and size may be necessary to accommodate the unique needs of each patient. Furthermore, achieving effective treatment for many diseases often requires sustained and precise release of high concentrations of minocycline.

Gelatin methacryloyl (GelMA) is a commonly used bioink due to its excellent printability, biocompatibility, biodegradability, and ease of modification. GelMA is photocrosslinkable with tunable porosity, swelling ratio and stiffness, making it an ideal candidate for encapsulating therapeutic agents. Previous studies have reported on the direct encapsulation of minocycline in GelMA scaffolds for sustained release. However, during the photopolymerization process of GelMA, free radicals can potentially oxidize the minocycline loaded into the bioink, thereby compromising its efficacy.

In this study, we utilized an ion-pairing mechanism between MH, dextran sulfate (DS), and magnesium to form nanoparticle complexes (MH-DS) for encapsulating and protecting MH. The nanoparticles were subsequently loaded into different batches of GelMA and 3D printed into various scaffold modalities for controlled release. To evaluate the anti-oxidative properties of the released MH from the printed scaffold, an in vitro oxidative stress model with L929 fibroblasts was employed.


Materials and Methods:: GelMA with 90% methacrylation was used in this study. To prepare MH-DS complexes, 2.4 mg/mL dextran sulfate (dissolved in 14.4 mM magnesium chloride) was mixed with 2 mg/mL minocycline, followed by vortexing for 10 seconds (Fig. 1A). The ink for printing was prepared by dissolving GelMA and 0.2% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, photoinitiator) in the solution with MH-DS complexes. Scanning Electron Microscopy (Zeiss Supra) was used to visualize nanoparticle complexes in lyophilized GelMA (Fig. 1B). The in vitro release behavior of MH-DS complexes in GelMA was evaluated by printing the ink into a scaffold with an extrusion-based bioprinter (SunP Biotech). An oxidative stress-induced apoptotic model (hydrogen peroxide, 1000 mM) was created by printing a cell-laden (fibroblast L929, 3 million/ml) GelMA scaffold covered by a layer of MH-DS complexes as a topical dressing (Fig. 1D). After incubation overnight, cell survival rate was measured using the AlamarBlue assay (Thermo Fisher).

Results, Conclusions, and Discussions:: The SEM images revealed that the MH-DS complexes are composed of nanoscale particles aggregated into a cluster and were encapsulated in the GelMA network (Fig. 1B). The release of MH from MH-DS in GelMA was sustained and steady over a week, with a 28.2±1.6% burst on the first day. In contrast, the control group, which used MH directly mixed with GelMA, had a 33.4±2.1% burst on the first day and stopped releasing on day 4. The overall accumulated release of the MH-DS group was 71±3.1%, while that of the control group was only 43.4±1.1%, indicating that free radicals during crosslinking compromised the MH (Fig. 1C). The L929 cell viability after oxidative stress induced by 1000 mM hydrogen peroxide was 48±10.9% (positive control), while the MH-DS complexes with a 0.375 mg/mL MH loading had the highest viability at 71.6±9.8% (p=0.0401). Other MH loading concentrations did not significantly differ from the positive control group (Fig. 1E, 1F).

We demonstrate the successful incorporation of MH-DS nanoparticle complexes into GelMA for achieving controlled drug release in bioprinted scaffolds. The released MH at specific concentrations exhibited significant protection to fibroblasts under oxidative stress. Our approach shows great promise as a versatile platform for various applications, including 3D printed wound dressings, implantable scaffolds, in vitro disease studies.



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