Direct Cryo Writing of Aerogels via 3D Printing of Aligned Cellulose Nanocrystals Inspired by the Plant Cell Wall

Round 1

Reviewer 1 Report

The research is novel and interesting. The manuscript is well organized and well written. Using different ratio of cellulose nanocrystals and xyloglucan bioinks to 3D print while freezing is an interesting approach. Figures and graphs are legible.

Introduction is very clear giving sufficient information regarding all aspects of the paper. However, few more reference regarding bioprinting can be included.

Figure 1 (b & c) needs scale bars

Methods section needs to include statistics sub section for describing statistical analysis of rheology, mechanical testing and 3D printing 

Results could use sub-sections similar to that of methods section

Author Response

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Reviewer 2 Report

The authors combine 3D printing (direct writing) and freeze casting process to fabricate multi-scale objects mimicking the organization in the plant cell wall. The alignment of the resulted aerogel object is generated from the freeze casting. While the paper is well written and organized, and the proposed idea is technically sound, but the proposed idea is not new. In Line 100, the authors claim “To the best of our knowledge, this is the first report of 3D printing of objects with aligned nanoparticles via the freeze casting process.” In fact, using 3D printing and freeze casting process to achieve aligned nanoparticles have been explored by some research groups. For example, the paper “Biomimetic 3D Printing of Hierarchical and Interconnected Porous Hydroxyapatite Structures with High Mechanical Strength for Bone Cell Culture” studied exactly the same approach and addressed the same issues (such as shear thinning properties, aligned microscale structures, mechanical properties etc.). The same approaches have been used to 3D print graphene aerogel nano materials and ceramic (silica, alumina) aerogel nano materials. The authors are suggested to conduct even more extensive review and identify the main contribution. In line 348: the authors claim “printing free standing in air without the typical requirement to use an additional a support material, it is not clear how this can be achieved. Compared with other extrusion approach such as direct writing and fused deposition modeling, the freezing printing approach does not seem to have special mechanism to print free standing features. As the layers grow up, the effect of the cold plate (thermoelectric cooler) is lower due to the low conductivity of the frozen ice, how to properly freeze the high parts? Since the material can be printed under certain shear-thinning conditions, there is no need to freeze the material during the printing, which complicates the process. Instead, the part can be printed in room temperature and the printed part can be frozen and freeze dried afterward, it would be interesting to compare the microstructures obtained by these two approaches.

Author Response

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Reviewer 3 Report

Doron Kam et al design a porous structure by combing 3D printing with liquid nitrogen. The ink viscosity, mechanical strength, and porous structure are characterized. Overall, the study is interesting. 

The weakness of the work is poor mechanical strength. The authors want to mimic the wood cell wall. But note that the mechanical properties of the wood cell wall are much stronger than the mechanical strength obtained here. Based on the mechanical strength, what is the significance of this structure?

The second weakness is this work is lack of demonstration of application. 

Author Response

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Round 2

Reviewer 2 Report

The reviewer appreciates the authors acknowledge the related work and reclaim the contribution of feedback-based temperature control for ice formation, though the reviewer still thinks the idea of combining 3D printing and freeze casting is not the major contribution. The freeze casting is spontaneously induced under the gradient temperature during freeze printing even without precise temperature control. In the Zhang work, the authors mentioned “by integrating 3D printing ice and freeze casting to print GA“ at the beginning of the second paragraph. The alignment of the 2D materials (graphene sheet) is also shown. Therefore, the reviewer suggests the authors revise some of the statements such as “This process is different than the freeze casting and printing under the feedback-controlled freezing process used herein, which leads to alignment of the dispersed particles”. 

The reviewer still have concerns regarding the questions in the first review: (1) line 365: “printing “free standing in air”, the reviewer is still not clear how the authors handle the free standing features, such as overhang structure where the material cannot be printed in air without support structures; (2) the authors used a standard thermoelectric cooler (TEC), what is the lowest temperature it can provide? As the part grows up, the cold conduction will be very inefficient as the printing material has relatively low thermal conductivity, the top layer may not reach freezing point. Did the printing process pause between layers to allow the top layer temperature reaches freezing point? Did the author control the temperature by radiation? such as the refrigerator as the authors reviewed. It is very helpful if the authors provide more details to allow the community to follow and duplicate the results; (3) since the feedback-based temperature control is part of the major contributions of this papers, the reviewer suggest the authors add more details on how the temperature control was achieved by the thermal imaging and control (PID?), and how the authors validate the consistent porous structure resulted from the consistent temperature gradient by the feed-back control.

Author Response

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