3D Printing of a Hyaluronan Bioink With Double Ge-lation Mechanism for Independent Tuning of Shear-Thin-ning and Shape Fixation

Introduction: Biomedical 3D printing is emerging as an indis-pensable tool for the fabrication of tissue-like constructs. Extru-sion-based bioprinting needs bioinks capable of low-shear ex-trusion, shape retention, adequate cell viability and composition similar to native tissues. In this study, we describe a tyramine-modified hyaluronic acid (THA) where the shear-thinning prop-erties can be tuned independently of the final curing step owing to a double gelation mechanism. The relation between printabil-ity and rheological properties was determined, as well as their variations upon cell addition and medium modification.Materials ans Methods: THA was synthesized by grafting tyramine to hyaluronan via amide formation in water achiev-ing 15.5% substitution degree. THA was dissolved at final con-centration of 2.5% w/v and added to 1) horseradish peroxidase and low concentration of H2O2 for enzymatic crosslinking, and 2) eosin Y (EO) for green light-triggered crosslinking. Rheo-logical properties were measured with an Anton Paar MCR302 Rheometer. hMSCs from bone marrow; bovine chondrocytes from fetlock joints and hTERT fibroblasts were laden in the bioink. A bioprinter RegenHU 3D Discovery® was used to print over a range of nozzle geometries, pressures, and light exposures. Cell viability and its variation upon medium compo-sition and printing conditions was determined with live/dead, CellTiter-Blue® and trypan blue assays in triplicates.Results: The attributes of the THA bioink here presented are: i) single component but 2 distinct crosslinking mechanisms, allowing ii) tuning of shear-thinning independently of the post-printing curing; iii) no sacrificial components or rheologi-cal additives; iv) curing with visible light for shape stability; v) possibility to post functionalize; vi) preservation of hyalu-ronan structure owing to low modification degree. Enzymatic crosslinking of THA to a low controlled extent produced a soft shear-thinning gel with viscosity between 6 and 25 Pa•s at the shear rates experienced during extrusion, and therefore suitable for low-shear extrusion and consequently for cell encapsula-tion. Cell addition during enzymatic crosslinking revealed a dose-dependent decrease in viscoelasticity, however the effect was compensated by increasing the levels of H2O2 in the precur-sor preserving viability. Crosslinking with light did not produce this effect, and therefore the cell-induced decrease in viscoe-lasticity was putatively attributed to cell uptake of H2O2, even though at the ppm-range employed the H2O2 did not induce any cell death.In PBS medium, EO displayed a dose-dependent cytotoxicity, likely due EO endocytosis. Cell death was significantly reduced at EO concentration below 0.02% and by adding 10% foetal bovine serum in the medium.Cell-laden criss-cross constructs with high shape fidelity were printed, displaying 24 hours viability up to 93% for all cell types. Viability was maintained or increased after 14 days in culture.Generally, printing fidelity and accuracy in biofabrication are still poorly defined and depend critically on the printer and the environmental conditions. We have identified the damping fac-tor = G /G as measurable and reliable printability index, with the best results achieved with values between 0.4 and 0.6. The bioink was too fluid for values above and granular and poorly extrudable for values below this range. The damping factor is easily quantifiable, and therefore an absolute magnitude com-parable objectively across laboratories to evaluate the printabil-ity of bioinks.Conclusion: We have introduced a bioink based on hyaluronan with a double gelation mechanism for independent tuning of shear-thinning and final curing with visible light. These addi-tional features allowed accurate tuning of the bioink properties, without additives. The damping factor was identified as reliable printability predictor.