Swelling characteristics and biocompatibility of ionic liquid based hydrogels for biomedical applications

Polymers are commonly used in medical device manufacturing, e.g. for drug delivery systems, bone substitutes and stent coatings. Especially hydrogels exhibit very promising properties in this field. Hence, the development of new hydrogel systems for customized application is of great interest, especially regarding the swelling behavior and mechanical properties as well as the biocompatibility. The aim of this work was the preparation and investigation of various polyelectrolyte and poly-ionic liquid based hydrogels accessible by radical polymerization. The obtained polymers were covalently crosslinked with N,Nʼ-methylenebisacrylamide (MBAA) or different lengths of poly(ethyleneglycol)diacrylate (PEGDA). The effect of different crosslinker-to-monomer ratios has been examined. In addition to the compression curves and the maximum degree of swelling, the biocompatibility with L929 mouse fibroblasts of these materials was determined in direct cell seeding experiments and the outcome for the different hydrogels was compared.

3. Line 112: How much hydrogel (or how many milliliters) was added between two 50 mm diameter cover glass for the biocompatibility assay?
Response from the authors: The information has been added to the materials and methods part of the manuscript (line 112): "For biocompatibility testing, 1 mL of the monomercrosslinker solution was polymerized between two 50 mm diameter cover slips to obtain specimen with the maximum possible surface." 4. Clearly explain how the dead cells were quantified? and compared with which CONTROL to calculate relative cell viability (Fig. 6).
Response from the authors: Thank you for this hint. We extended the legend of Figure  Here, the poor cell counts can be result from two reasons: (1) Lower cellular adhesion property of the hydrogel itself (due to difference in porosity or swelling, as discussed by the authors) and/or (2) Hydrogel induced cytotoxicity that allows cells to initially attach on the hydrogel surface, BUT due to the cytotoxic hydrogel components, cells undergo apoptosis and finally detach from the hydrogel surface. To prove that the hydrogel ITSELF is not cytotoxic, it is important to perform ethidium homodimer-1 (EtHD-1) staining along with calcein-AM. EtHD-1 can detect the hydrogel-attached apoptotic cells to determine whether hydrogel itself is cytotoxic or not. Please address and discuss this issue.
Response from the authors: Thank you very much for your detailed comment. Our microscopic analysis did not aim to provide a quantitative conclusion on cytotoxicity. Rather, it was intended to support the quantitative analysis with the CellQuanti Blue Assay. The microscopic images should prove that the cells were actually actively spreading on the hydrogel, displaying another quality of the hydrogels besides cell viability. In earlier experiments using a different preparation methods of the specimens, we found that the cells slipped off the hydrogel and grew on the cell culture polystyrene. Very good cell viability could be measured here as well.
Furthermore, we were able to make a qualitative evaluation of the cell morphology by microscopic analysis. The Calcein AM staining was also intended to demonstrate that the cells on the hydrogels are viable, but not to provide a quantification for the evaluation of cytotoxicity. The question of whether apoptotic or possibly also necrotic processes result in a reduction of cell count and cell viability is undoubtedly very interesting, but not the subject of this study. Rather, we intended to pre-select candidates from a whole range of hydrogels based on basic parameters. After selecting generally suitable hydrogels for specific biomedical applications, this materials should indeed be tested for their potential to induce apoptosis. We added another paragraph to our manuscript (line 293-294): "To select suitable hydrogels for specific biomedical applications, this materials should additionally be tested for their potential to induce apoptosis." 6. No information regarding degradation rate of these hydrogel is provided. Since these hydrogels are proposed to be used for biomedical applications, it is very critical to study their rate of degradation.
Response from the authors: The degradation of these hydrogels is certainly interesting and necessary for this type of application. We added a paragraph to our manuscript regarding this topic (line 285-290): "However, when investigating materials for an application of the body, not only direct biocompatibility has to be ensured, but in addition possible toxic or harmful effects of degradation products have to be taken into account. The hydrogel materials in the presented work are based on functionalized methacrylate monomers crosslinked with MBAA. This compound class has shown no or little cytotoxic degradation products in literature (44)(45)(46). However, since different functionalities such as sulfonic acid or amino groups may have a huge impact on the biocompatibility, especially of degradation products, this behavior has to be investigated before application testing in vivo and regarding potential approval processes for biomedical uses." However, these experiments and results would go beyond the scope of this manuscript but will be addressed in the ongoing work on this topic. 7. Please include discussion regarding the immune cell response (especially macrophages) toward these hydrogels if previously reported in the literature. Importantly, discuss whether the degradation products of these hydrogel can induce severe immune response.

Response from the authors:
Thank you for this interesting hint. In our response to comment 5 we already claimed that we intended to pre-select hydrogels for specific biomedical applications based on basic parameters. Nevertheless, aspects of the immunotoxicity are very interesting and must be addressed within approval process for biomedical applications. Indeed, we found evidence for inflammatory processes in vitro and in vivo caused by hydrogels in literature. For example Amer et al., 2019 found an Toll-like receptor mediated activation of macrophages on synthetic poly (ethylene glycol) hydrogels. We appreciate your advice and will consider this in our future studies. We added a paragraph to our manuscript regarding this topic (line 290 -293): "Another aspect for potential approval processes is the immunotoxicity of the body to different kind of materials. In literature we found evidence for inflammatory processes in vitro and in vivo caused by hydrogels. Amer et al. reported about an toll-like receptor mediated activation of macrophages on synthetic poly (ethylene glycol) hydrogels (47)."