Patients requiring soft tissue reconstruction caused by defects or pathology may require biomaterials that provide a void volume for subsequent vascularization and new tissue formation, as autografts are not always a viable option. Here supramolecular hydrogels represent promising candidates due to their 3D structure being similar to the native extracellular matrix and their cell entrapment capability. Over recent years, guanosine (Guo)-based hydrogels have increasingly emerged, in which the nucleoside self-assembles into ordered structures (G4-quadruplex) and ultimately into nanofibrillar networks by the π-π stacking of G-quartets and coordination of central K+ ions. While such hydrogels typically exhibit a short lifetime, the use of boronic acid (BA) significantly enhances their stability. The aim of the present study was to combine this technology with 3D bioprinting to produce binary cell-laden hydrogels consisting of Guo and guanosine 5-monophosphate (GMP) stabilized by BA and K+, and to optimize printability and the survival of the entrapped cells. Such a system would then allow to tailor the biomaterial to the respective soft tissue defect and thus improve tissue reconstruction.
Various compositions of Guo (10-120 mM), GMP (10-120 mM), BA (10-60 mM) and KOH (10-60 mM) were mixed at 80 °C, slowly cooled down, and assessed for gelation by inversion test. Printability was subsequently evaluated by a semi-quantitative filament collapse and fusion test. The very best hydrogel composition was then immersed in a hyperbranched poly(ethylenimine) (PEI) solution (5 mgmL-1, 15min) and characterized by scanning electron microscopy (SEM) and rheological studies (strain sweep, dynamic step-strain sweep and peak-hold assay). Furthermore, nutrient permeability (FITC-Dextran) and hydrogel stability (immersion in complete medium at 37 °C) were determined. Finally, rat mesenchymal cells (rMCs) were entrapped in the hydrogels and studied for 21 days after printing, including cell viability and morphology assessment by confocal laser scanning microscopy (CLSM), and monitoring of the adipogenic differentiation (Oil-red O solution).
From 49 hydrogel compositions that passed the inversion test, 15 exhibited suitable 3D printing properties. To improve long-term stability, hydrogels were subsequently treated with PEI, and the best composition was analyzed by SEM, showing nanofibrillar structures evident of successful G4-quadruplex formation. Furthermore, rheological analysis revealed good printing and thixotropic properties, while successful diffusion of FITC-dextran molecules (70, 500 and 2000 kDa) into the hydrogel confirmed that nutrients of various sizes may diffuse through the scaffold. Finally, a cell viability of 85% after 21 days was observed but with exclusively rounded morphology. However, lipid droplets were identified after 7 days, indicating cell functionality and successful differentiation under adipogenic conditions.
Our results demonstrate that printed Guo/GMP hydrogels exhibit extensive nanofibrillar networks and good printability and thixotropic properties. Stability was verified for 21 days in medium, and embedded cells showed good survival despite rounded morphology. Under adipogenic conditions, lipid droplets were observed, witnessing successful differentiation and functionality of the entrapped cells. Due to the demonstrated bioprintability, our Guo/GMP hydrogels may hold great potential for the reconstruction of soft tissues."