INTRODUCTION:Enzymatically mediated crosslinked hyaluronan-tyramine hydrogels (HA-Tyr) are promising matrices for tissue engineering and regenerative medicine. However, due to relatively low tyramine modifications of the hyaluronan backbone achieved, HA-Tyr matrices have weak and a narrow range of mechanical properties. Therefore, we report here the use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) coupling agent for the iterative grafting of tyramine onto hyaluronan and fabrication of modular hyaluronan cellular microenviroments. MATERIALS AND METHODS: HA-Tyr was prepared by DMTMM mediated amidation and the stepwise reactions perfomed repeating the same protocol. HA-Tyr conjugates were characterized using UV-vis and 1H NMR, degradation by weight loss measurement in presence of hyaluronidase and Young's modulus in an unconfined compression stress-relaxation set-up. hMSCs were isolated from human bone marrow and embedded in HA-tyr at 5x106 cells per ml and cultured in DMEM. Cell viability and cell spreading in hydrogels were assessed by live-dead staining. RESULTS: The final yields of substituted Tyr (DS) on HA were 6.5±0.5% at 24 h (single addition), 15±1% at 48 h (two step reaction), and increased up to DS of 20±1% and 20±1.5% at 72 h and 96 h (three and four step reactions) with similar molar ration of reactants. EDC/NHS stepwise reactions lead to unsoluble HA-Tyr due to build-up of by-products and unspecific crosslinks. At low H2O2 concentration (0.68 mM) the E of formed enzymatic HA-tyr hydrogel is independent of the DS, whereas for 1.1 mM H2O2, E increased with DS. hMSCs in all conditions showed high viability (85-98%) over 6 days as quantified with Live/Dead staining and spreading could be observed in hydrogels with E lower than 10 kPa at 6 days. DISCUSSION and CONCLUSION: The iterative use of DMTMM as coupling agent increased the yields of tyramine functionalization, which was reflected by a two-fold increase in HA-Tyr hydrogels E. Viable encapsulation of human mesenchymal stem cells was achieved in all hydrogels and distinct cellular spreading observed in the absence of additional binding cues. The tunable HA-Tyr hydrogels are optimized to study a wide range of cellular behavior.