Development of Injectable Thermosensitive Chitosan-Based Hydrogels for Cell Encapsulation

Round 1

Reviewer 1 Report

Preparation and investigation of injectable, thermoresponsive and biocompatible chitosan (CH) based hydrogels are discussed in the article. This type of materials corresponds to very interesting and challenging scientific field as drugs or live cells carriers and storages for different medical and biochemical aims.

Authors applied in their work CH because this biopolymer shows well-known biocompatibility, low toxicity and degradability. They claim that combined for the first time CH with sodium hydrogen carbonate (SHC) solution as gelation agent and studied the influence of the beta-glycerol-phosphate (BGP) and L-arginine (ARG) addition on the CH+SHC hydrogel properties.

It confirmed experimentally that all CH+SHC hydrogels are prepared in few minutes at 37°C. Addition of BGP and ARG didn’t influence essentially on jellification process. All prepared hydrogels presented lower swelling rate in comparison with pure CH. Authors associated this fact with interaction of additional components with biopolymer macromolecules. Prepared hydrogels were stable in biological liquids and mechanically strong. Preliminary biological tests showed high biocompatibility of all hydrogel systems because they don’t release any cytotoxic substances. Human neuroblastoma cells SH-SY5Y were encapsulated within the hydrogel and distributed uniformly in its volume.

It would be beneficial for the manuscript to discuss rheological properties of such difficult multicomponent viscoelastic systems. Different rheological approaches usually give a lot of information about mechanical properties or stability and help to discuss the mechanism of jellification.

In my opinion the manuscript is well organized and scientifically qualified and could be published after minor revision.

Author Response

Response to Reviewer 1 Comments

Point 1: It would be beneficial for the manuscript to discuss rheological properties of such difficult multicomponent viscoelastic systems. Different rheological approaches usually give a lot of information about mechanical properties or stability and help to discuss the mechanism of jellification.

Response 1: We thank you the Reviewer for the positive overall evaluation of our manuscript. We agree with the reviewer that rheological properties are important for hydrogel systems, therefore we performed different rheological tests both at 21°C (room temperature) and 37°C (body temperature) in order to evaluate the storage modulus G' and the loss modulus G'' at the two relevant temperatures. The rheological test conditions and results have been added in the SI and mentioned in the manuscript (see text in red). Briefly, G' is higher than G'' for all hydrogel formulations at both 21 and 37°C, indicating that the elastic contribution is prevalent on the viscous one (material already at the gel state and not sol anymore); however, both G' and G'' are significantly higher when measured at 37°C confirming the thermoresponsive behaviour observed in the inversion tube test.

Reviewer 2 Report

The manuscript entitled “Development id injectable thermosensitive chitosan-based hydrogels for cell encapsulation” presents an interesting work on the fabrication of thermosensitive chitosan-based hydrogels using a combination of organic and inorganic salts for the encapsulation of the cells. The article is well written and discussed, demonstrating a systematic approach and explanation of the observations. I recommend publishing this manuscript in the present form.

Author Response

Response to Reviewer 2 Comments

Comments: The manuscript entitled “Development id injectable thermosensitive chitosan-based hydrogels for cell encapsulation” presents an interesting work on the fabrication of thermosensitive chitosan-based hydrogels using a combination of organic and inorganic salts for the encapsulation of the cells. The article is well written and discussed, demonstrating a systematic approach and explanation of the observations. I recommend publishing this manuscript in the present form.

Response: We thank very much the Reviewer for the positive evaluation of our manuscript.

Reviewer 3 Report

The manuscript reported three formulations of thermosensitive chitosan hydrogels and their characterizations on physical properties and biocompatibility. There are a few suggestions.

1. The results seemed that the addition of ARG into the hydrogel did not perform much improvement for cellular encapsulation. What is the purpose to add ARG into the hydrogel system? Please supply it in the Introduction part to make the manuscript more readable.
2. Rheological analysis should be performed to indicate the accurate gelation time under 37℃ and the mechanical properties (storage modulus and loss modulus) after gelation.
3. Live/dead staining on the cells encapsulated in the hydrogels should be conducted to directly indicate the cellular toxicity. Additionally, a conducive cellular environment always can support cell growth, so cellular proliferation tests on these hydrogels are also necessary to prove these hydrogels are applicable for cellular encapsulation.

Author Response

Response to Reviewer 3 Comments

Point 1: The results seemed that the addition of ARG into the hydrogel did not perform much improvement for cellular encapsulation. What is the purpose to add ARG into the hydrogel system? Please supply it in the Introduction part to make the manuscript more readable.

Answer 1: We thank the Reviewer for her/his useful comment and question. Indeed, the introduction of ARG did not seem to lead to much improvement on the physical (swelling, stability, mechanical) properties of the hydrogel in the reported sudies. The rationale behind ARG introduction rose from our previous studies where ARG was able to improve the in vitro stability of chitosan 3D structures, such as scaffolds and electrospun membranes [1-3]. In the present work, we aimed at verifying if ARG could give a similar contribution even in chitosan hydrogel systems. L-Arg is a conditionally essential amino acid, involved as a precursor in many important biochemical pathways in cellular physiology (i.e. nitric oxide pathway involved in nerve regeneration) [4-6], and therefore an enhancer of some key cell processes, e.g. collagen synthesis, T-cell mediated responses, and also the release of pituitary hormones [7]. For these reasons we used it as a non-toxic ionic cross-linking biomocelule for chitosan structures, hopefully eliciting enhanced biological responses.

1. Izzo, D.; Palazzo, B.; Scalera, F.; Gullotta, F.; Lapesa, V. .; Scialla, S.; Sannino, A.; Gervaso, F. Chitosan scaffolds for cartilage regeneration: influence of different ionic crosslinkers on biomaterial properties, INT J POLYM MATER PO 2019, 68 (15), 936–945.
2. Scialla, S.; Barca, A.; Palazzo, B.; D’Amora, U.; Russo, T.; Gloria, A.; De Santis, R.; Verri, T.; Sannino, A.; Ambrosio, L.; Gervaso, F. Bioactive Chitosan-Based Scaffolds with Improved Properties Induced by Dextran-Grafted Nano-Maghemite and l-Arginine Amino Acid. J. Biomed. Mater. Res. - Part A 2019, 107 (6), 1244–1252.
3. Nitti, P.; Gallo, N.; Palazzo, B.; Sannino, A.; Polini, A.; Verri, T.; Barca, A.; Gervaso, F. Effect of L-Arginine treatment on the in vitro stability of electrospun aligned chitosan nanofiber mats. Polymer Testing 2020, 91, 106758.
4. Greene, J.; Feugang, J.; Pfeiffer, K.; Stokes, J.; Bowers, S.; Ryan, P. L-arginine enhances cell proliferation and reduces apoptosis in human endometrial RL95-2 cells. Reprod. Biol. Endocrinol. 2013, 11 (15), 1-11.
5. Racké, K.; Warnken, M. L-arginine metabolic pathways, Open Nitric Oxide J. 2010, 2, 9–19.
6. Damodaran, V.B.; Bhatnagar, H.; Rubin, D.; Reynolds, M.M. Chapter 6 - nitric oxide donors in nerve regeneration, in: A.B. Seabra (Ed.), Nitric Oxide Donors, Academic Press, 2017, 141–168.
7. Shi, H.P.; Wang, S.M.; Zhang, G.X.; Zhang, Y.J.; Barbul, A. Supplemental L-arginine enhances wound healing following trauma/hemorrhagic shock, Wound Repair Regen. 2007, 15 (1), 66–70.

According to the Reviewer suggestion we added the above reported considerations and comments in the Introduction part of the revised manuscript (text in red). The references in the manuscript have also been update accordingly.

Point 2: Rheological analysis should be performed to indicate the accurate gelation time under 37℃ and the mechanical properties (storage modulus and loss modulus) after gelation.

Answer 2: We thank you the Reviewer for the valuable suggestion. We agree with the reviewer that rheological properties are important for hydrogel systems, therefore we performed different rheological tests both at 21°C (room temperature) and 37°C (body temperature) in order to evaluate the storage modulus G' and the loss modulus G'' at the two relevant temperatures. The rheological test conditions and results have been added in the SI and mentioned in the manuscript (see text in red). Briefly, G' is higher than G'' for all hydrogel formulations at both 21 and 37°C, indicating that the elastic contribution is prevalent on the viscous one (material already at the gel state and not sol anymore); however, both G' and G'' are significantly higher when measured at 37°C confirming the thermoresponsive behaviour observed in the inversion tube test.

Point 3: Live/dead staining on the cells encapsulated in the hydrogels should be conducted to directly indicate the cellular toxicity. Additionally, a conducive cellular environment always can support cell growth, so cellular proliferation tests on these hydrogels are also necessary to prove these hydrogels are applicable for cellular encapsulation.

Answer 3: We thank the Reviewer for her/his useful comments. Following the suggestions, live/dead staining was performed on cell-laden hydrogels. Accordingly, new section have been added in the Materials and methods (2.8) and the Results (3.7), respectively. We strongly agree that a proliferation test would also provide useful indications on the developed hydrogels. Considering that in this manuscript we focused on the development of the hydrogel systems and their preliminary biological assessment, further tests will be performed on a selected hydrogel formulation.