(395f) Nanoengineered Granular Bioinks: Overcoming the Limitations of 3D Bioprinting of Granular Hydrogels

Introduction: The field of tissue engineering has benefited from the development of three-dimensional (3D) bioprinting technology as well as granular hydrogel scaffolds made up of hydrogel microparticles (microgels). Commonly, microgel suspensions are 3D printable when they are tightly packed. However, tightly packed microgels in single-component granular hydrogel inks undergo significant interstitial void space reduction, compromising scaffold porosity. This work introduces the first class of nanoengineered granular bioinks (NGB), which overcome the limitations of single-component granular hydrogel inks, i.e., attaining porosity while enabling high extrudability and printability.

Materials and methods: For NGB preparation, interparticle interactions were engineered via the reversible self-assembly of heterogeneously charged colloidal nanoclay adsorbed onto the microgels.^[1] Gelatin methacryloyl (GelMA) was synthesized using a standard protocol.^[2] Briefly, 20 g of gelatin was dissolved in 200 mL of Dulbecco's phosphate-buffered saline (DPBS), followed by the addition of 16 mL of methacrylic anhydride at 50 °C. The reaction was stopped after 2 h by adding an excessive amount of DPBS (400 mL). The solution was then dialyzed against ultra-pure water for 10 days, followed by lyophilization to yield solid GelMA. A high-throughput step-emulsification microfluidic device was used to fabricate monodispersed GelMA droplets. A 10% w/v GelMA solution in DPBS, including 0.1% w/v of a photoinitiator, was used as the aqueous phase, while the Novec 7500 engineering fluid, including a biocompatible surfactant (2% v/v), was used as the oil phase. GelMA droplets were stored at 4 °C for physical crosslinking, and an optimized concentration of nanoclay suspension (LAPONITE XLG) was added to the microgel suspension, yielding NGB. To investigate porosity, the diffusion path of a high molecular weight fluorescent dye (fluorescein isothiocyanate (FITC)-dextran, Mw ~ 2 MDa,) was imaged using a fluorescence microscope. The immediate penetration of NIH/3T3 fibroblast cells was shown via topical seeding.

Results and discussion: NGB had excellent printability with high resolution, shape fidelity, and structural integrity, as shown in Figure 1A-B. The rheological properties of NGB were assessed using steady shear sweep and oscillatory recovery tests (Figure 1C), showing that NGB is a shear-thinning bioink, undergoing proper recovery when alternating high or low strains was applied. The hanging filament length (L_f) test revealed that NGB had a significantly higher L_f than the jammed (tightly packed) bioink, indicating its superior extrudability (Figure 1D). The FITC-dextran diffusion in NGB and loosely packed (unjammed) scaffolds occurred rapidly, unlike bulk or tightly packed granular inks, as shown in Figure 1E(i). Topical seeding of fibroblast cells showed high cell penetration in the NGB and loosely packed (control) scaffolds, while limited penetration was observed in the jammed scaffold, and no cell infiltration was yielded in the bulk scaffold (Figure 1E(ii)). Overall, the results highlight the capability of NGB for the 3D printing of tissue engineering scaffolds with high shape fidelity, structural integrity, and preserved interconnected porosity.

Conclusions: In summary, the NGB attains preserved porosity while enabling high extrudability and printability, rendering it a unique and versatile platform for the biofabrication of tissue engineering scaffolds. This work presents a step forward in the field of 3D bioprinting of granular scaffolds and highlights the promise of NGB as a bioink for a wide range of tissue engineering applications. Future research will focus on optimizing the properties of NGB for specific tissue types, as well as exploring its potential for creating multi-layered and multi-functional structures.

References:

[1] Z. Ataie, S. Kheirabadi, J. W. Zhang, A. Kedzierski, C. Petrosky, R. Jiang, C. Vollberg, A. Sheikhi, Small 2022, 18, 2202390.

[2] Z. Ataie, A. Jaberi, S. Kheirabadi, A. Risbud, A. Sheikhi, J. Vis. Exp. 2022, 190, e64829.