Poster O16 - NGB II: A Thermostable Nanoengineered Granular Bioink for In Situ Biofabrication

Introduction: Gelatin methacryloyl (GelMA) is a widely used biomaterial to partially mimic the extracellular matrix (ECM), promote cell activities, and enable tissue regeneration. While its crosslinkable functional groups offer adjustable mechanical properties, the bulk GelMA hydrogel has challenges for biofabrication due to the coupling of stiffness and porosity: as the hydrogel stiffness increases to enhance printability, the nanoscale porosity decreases along with cell viability. To address this challenge, GelMA hydrogel microparticles (microgels) may be used as granular bioink building blocks to improve cell infiltration and viability because of the microscale porosity yielded from their assembly. However, physically crosslinked GelMA microgels are thermosensitive, limiting their potential for in situ fabrication at the physiological temperature (37 °C). Our first version of the bioink (NGB I) used these GelMA microgels as building blocks paired with heterogeneously charged nanoparticles imparting reversible self-assembly. The nanoparticles enhanced microgel-microgel interactions through electrostatic interactions, yielding shear thinning behavior. Although NGB I had the ability to print constructs with high shape fidelity and well-preserved interconnect pores, the low-temperature requirement limited the use of this bioink for in situ biofabrication. Incorporating thermostable GelMA microgels can mitigate this challenge to achieve a thermostable NGB (NGB II).

Materials and

Methods: GelMA was synthesized by dissolving 20 g of gelatin in 200 mL of Dulbecco’s phosphate buffered saline (DPBS), to which 16 mL of methacrylic anhydride was incrementally added at 50 °C. The reaction was terminated after 2 h by introducing an excess of DPBS (400 mL), and the product was dialyzed against ultra-pure water for 10 days, followed by lyophilization and storage. For droplet formation, a high-throughput step-emulsification microfluidic device was used, enabling generation of over 40 million droplets per hour. GelMA solution (10 wt.% in DPBS with 0.1 wt.% photoinitiator) served as the aqueous phase and Novec 7500 oil containing 2% v/v surfactant was used as the oil phase. Subsequent photocrosslinking was performed at 15 mW cm-2 using a 395-400 nm light source for 10 min, deliberately leaving some methacryloyl groups unreacted. Post-crosslinking, 1 wt.% N-vinylcaprolactam and increased photoinitiator concentration to 0.3 wt.% were introduced to promote microgel covalent assembly using the residual MA groups. Nanoclay colloidal suspension (LAPONITE XLG) was added to the microgel suspension at concentrations of 0% (control), 1%, or 2% to yield different formulations of NGB II.

Results, Conclusions, and Discussions: NGBII shows filament formation capabilities as a result of nanoparticle incorporation. Filament formation is an essential characteristic for printable bioinks. Microgel suspensions without nanoparticle incorporation show no filament formation. NGB II also had rheological characteristics of shear-thinning and high recovery. Overall, NGB II retained the advantageous properties of NGB I, such as high printability and shape fidelity, and facilitated in situ scaffold formation at physiological temperatures. This advancement may expand the possibilities of bioprinting granular scaffolds beyond the traditional biofabrication window.

Acknowledgements (Optional):