Sequential gelation of tyramine-substituted hyaluronic acid hydrogels enhances mechanical integrity and cell viability

Abstract

Tyramine-substituted hyaluronic acid (HA-Tyr) hydrogels formed by the oxidative coupling reaction of hydrogen peroxide (HO) and horseradish peroxidase (HRP) have been used for cellular encapsulation and protein delivery. Crosslinking density and gelation time can be tuned by altering the HO and HRP concentrations. Previous studies using HA-Tyr constructs report significant mechanical degradation after 21 days of culture. In this work, exogenous supplementation of HRP after initial gelation resulted in superior mechanical properties in acellular hydrogels and improved viability and proliferation in cell-laden constructs. Swelling of the acellular hydrogels was prevented in the samples receiving exogenous HRP. Monolayer studies showed no negative effect of relevant HRP concentrations on the viability of human adipose-derived stem cells (hASCs) and improved the viability of hASCs cultured with HRP and HO compared to HO alone. Taken together, this study demonstrates that HA-Tyr hydrogel properties could be modified by exogenous supplementation of HRP to tune scaffold degradation and improve cell viability by mitigating the negative effects of oxidative stress.

Enzymatically crosslinked silk-hyaluronic acid hydrogels

“…Tyramine-substituted HA has been previously synthesized to provide a biocompatible hydrogel that can be enzymatically crosslinked with HRP and H 2 O 2 , under physiological conditions [20–23]. But these hydrogels still have limited mechanical properties with moduli ranging under 10 kPa [24] and degrade by more than half of their initial weight by 1 month in vivo [25]. Thus, HA hydrogels can benefit from a composite system that can offer enhanced mechanical properties and resistance to degradation.…”

“…Although this method can be carried out under physiological conditions, the hydrogels are often washed prior to injection to extract the not reacted crosslinking agents [36] which are cytotoxic [37, 38] and can lead to loss of cell and tissue functions [21]. On the other hand, the enzymatic crosslinking method, using HRP and H 2 O 2 , has shown minimal cytotoxicity with the in situ gelation of HA hydrogels [24, 25]. Therefore, enzymatically crosslinking silk and HA can provide an alternative, cytocompatible method of forming composite hydrogels, while providing a format to understand how polymer concentration affects hydrogel properties.…”

Articular Joint-Simulating Mechanical Load Activates Endogenous TGF-β in a Highly Cellularized Bioadhesive Hydrogel for Cartilage Repair

“…The minimum concentration of H 2 O 2 necessary to obtain physically stable cell-laden hydrogels was 0.15 mM, which resulted in cell viability .95% as quantified with live/dead staining images at days 1 and 14 of culture ( Figure 1A). Increasing the concentration of H 2 O 2 to 0.3 or 0.6 mM did not significantly decrease cell viability in the presence of 0.5-U/mL HRP, although 1 When low concentrations of H 2 O 2 (0.3 and 0.6 mM) were used, the G# was always \1000 Pa, independent of cell density. When the concentration of H 2 O 2 was increased up to 1.6 mM, increased cell density dramatically decreased the G#, from 3070.0 6 70.7 Pa with 2.5 3 10 6 cells/mL to 620.5 6 170.4 Pa with 20 3 10 6 cells/mL (Figure 2A).…”

“…8 In line with previous findings, the concentration of HRP did not significantly influence cell viability, as reported in adipose tissue-derived MSCs within HA-Tyr hydrogels obtained with a sequential gelation mechanism. 1 The presence of cells reduced the viscoelastic properties of the hydrogels: an effect that could be counteracted by increasing the concentration of H 2 O 2 but not HRP. The role of H 2 O 2 as a regulator of the mechanical strength of HA-Tyr hydrogels has been reported in cell-free hydrogels.…”

Phenol–Hyaluronic Acid Conjugates: Correlation of Oxidative Crosslinking Pathway and Adhesiveness

“…Each HA–Ca or HA–Ty hydrogel was obtained upon addition of HRP and H 2 O 2 into the modified HA solutions, causing di–catechol and di–tyramine crosslinking in each solution ( Figure 3 a) [ 25 , 26 ]. During such crosslinking reaction, di–catechol and di–tyramine bonds form the HA polymeric network (e.g., hydrogels) with different color appearances.…”