Injectable and biodegradable PEG-based Hydrogel for cardiac tissue engineering

Ischemic heart disease is caused by cardiomyocyte death under hypoxic conditions, followed by the formation of a dysfunctional fibrotic scar, populated by activated fibroblasts. The presence of a high amount of reactive oxygen species (ROS) post-injury may play a role in affecting cardiomyocyte metabolism and viability, leading to cell apoptosis. Based on recent findings, ROS releasing hydrogels at proper dose could be exploited to train cardiomyocytes, enhancing their cardioprotection mechanisms. In this work, in vivo-like ROS were generated in water-based polymer solutions by using atmospheric pressure plasma jet (APPJ) devices and were exploited as vehicles for the delivery of ROS. Photo-crosslinkable poly(ethylene)glycol triblock copolymer (tPEO)-based hydrogels are excellent candidates for tissue engineering (TE) applications due to their tunable mechanical properties and versatile morphologies. Moreover, we already demonstrated that APPJ treatment of tPEO hydrogel enhanced final modulus with minor degradation of the polymer backbone chemistry at high concentrations. Although tPEO hydrogels present suitable mechanical properties for cardiac TE, their non-biodegradability limits the design of clinically injectable biomimetic materials. In this work, we aim to synthesize a biodegradable tPEO (btPEO) polymer, for the development of injectable, biodegradable and cell-adhesive hydrogels in combination with ROS as a minimally invasive therapy enhancing cardioprotection. We first investigated the ROS dose concentration on the cell viability (resazurin assays, at days 1, 3 and 7) and gene expression (a-SMA, at day 7) of human adult cardiac fibroblasts (HCFs) post APPJ-activated cell culture media exposure during 24h. In a second step, we synthetized tPEO polymer using atom transfer radical polymerization chemistry and btPEO was obtained by introducing lactic ester groups to the initial tPEO. Then, ROS-loaded hydrogels were prepared by treating tPEO solution (up to 10 min) prior mixing with btPEO and gelatin or gelatin methacryloyl (GelMa) with proper ratio, concentration and crosslinking degree to approach the stiffness of healthy cardiac tissues (target stiffness of around 1-10 kPa). Afterwards, ROS-loaded hydrogels were used for HCFs encapsulation and culture by assessing their viability (live/dead assays) and proliferation (resazurin assay) as a function of ROS dose. We discriminate a link between APPJ treatment time, ROS generation and the HCFs biological effects. An inferior ROS dose limit concentration (around max 80 uM H2O2) by applying a short APPJ treatment time (< 1 min duration) provides cardioprotection of HCFs. At higher APPJ treatment time (from 1 min and concentration higher than100 uM H2O2), HCFs started to be activated into myofibroblasts compared to control conditions after 7 days of culture. ROS stability in media was monitored and could be stored in water-based solutions (over 7 days at 4 C). However, a fast and efficient release within 30 min from the hydrogels was obtained once immersed in media, with minor alteration of the physicochemical properties. Hydrogels were stable in physiological and cell culture media at 37C for up to 1 month and biodegradability increased by increasing btPEO content. In this work we developed an injectable and biodegradable PEO-based hydrogel, in combination with ROS from APPJ as a potential solution in cardiac tissue regeneration.