Solid matrix-assisted printing for three-dimensional structuring of a viscoelastic medium surface

Gluconacetobacter xylinus (G. xylinus) metabolism is activated by oxygen, which makes the formation of an air-medium interface critical. Here we report solid matrix-assisted 3D printing (SMAP) of an incubation medium surface and the 3D fabrication of bacterial cellulose (BC) hydrogels by in situ biosynthesis of G. xylinus. A printing matrix of polytetrafluoroethylene (PTFE) microparticles and a hydrogel ink containing an incubation medium, bacteria, and cellulose nanofibers (CNFs) are used in the SMAP process. The hydrogel ink can be printed in the solid matrix with control over the topology and dimensional stability. Furthermore, bioactive bacteria produce BC hydrogels at the surface of the medium due to the permeability of oxygen through the PTFE microparticle layer. The flexibility of the design is verified by fabricating complex 3D structures that were not reported previously. The resulting tubular BC structures suggest future biomedical applications, such as artificial blood vessels and engineered vascular tissue scaffolding. The fabrication of a versatile free-form structure of BC has been challenged due to restricted oxygen supplies at the medium and the dimensional instability of hydrogel printing. SMAP is a solution to the problem of fabricating free-form biopolymer structures, providing both printability and design diversity.

A minor issue in Line 30: "BC is a polysaccharide synthesized from Gluconacetobacter xylinus". Please note that many different microorganism strains also produce BC. IN passing, please note that authors refer to Acetobacter Xylinus in Fig. 1.
Reviewer #2 (Remarks to the Author): The authors reported a solid matrix-assisted 3D printing (SMAP) processing technique of an incubation medium surface for the 3D fabrication of bacterial cellulose hydrogel-structures. This technique would allow the preparation of tailored bacterial cellulose-based 3D structures. Some points should be addressed before publication: There is little information regarding the incubation time needed to produce these structures. Most samples were incubated for 7 days. In the case of the tubular structure, the thickness was evaluated and it is clear that after 8 days no more thickness can be achieved. However, the blood vessel sample was incubated for 6 days. In static conditions, BC is often incubated for 7-14 days. Please, add some comments regarding why the authors used 7 days for most samples and if the incubation time could be controlled by modifying the glucose content of the ink. The authors should provide information regarding the properties of the BC network (diameter and longitude of BC fibers, aspect ratio of BC fibers, mesh size of BC network). The BC network properties depend on the growing media and conditions. For instance, the BC diameter should be compared with reported diameters of BC-based structures prepared with other techniques. The authors should report if BC network properties (diameter, mesh size, etc.) are constant or depend on the structure they print. Regarding the rheological characterization of the BC-based hydrogels, the authors only presented strain sweeps. Why? Did the authors performed temperature and frequency sweeps? Please report the thickness of the samples used for tensile tests. Fig. 2b. Please indicate the symbols used for the storage modulus (G´) and for the loss modulus (G´´). This paper presents 3D fabrication of CM-CNF/BC hydrogels using in situ BC production of bacteria through the PTFE microbeads system, through which air can be supplied to the viscous CM-CNF/bacteria mixtures. The idea of producing the hydrogels are unique and interesting, and the manuscript has a high originality. However, technical repeatability by readers is not sufficient also in terms of science, which should be improved before publication. Details are as follows.
1) The local amount of BC production in the hydrogels may significantly be influenced by the airfeeding levels. The even air-feeding is required to prepare homogeneous hydrogel thicknesses or amounts in throughout the hydrogel parts. How the authors controlled the homogeneous air-feeding in the whole interfaces?
2) The information of the CM-CNF prepared in this study is insufficient. More detailed information should be addressed in Supporting Information, such as the yield of CM-pulp, the degree of substitution, molecular weights, the degree of nanofibrillation, etc., otherwise readers cannot repeat the experiment. The details of kraft (not craft) pulp also should be addressed, such as the hemicellulose content, degree of polymerization, etc. In general, dissolving pulps with high cellulose contents are used in carboxymethylation. However, in this study, the kraft pulp containing a significant amount of hemicelluloses was used. Please explain the reason.
3) In this study, amidation was used to fix collagen to BC. Were the EDC, NHS used in this study are really safe to be used as artificial blood vessels? The amidation used in this system is not simple, and some residual EDC or NHS may remain in the hydrogels as counterions of carboxy groups of CM-CNF. 4) How the authors removed the BC cells? The 0.1% NaOH post-treatment may have partly removed CM-CNF components.
Reviewer #4 (Remarks to the Author): The reported work attempts to mainly propose preparation of 3D ink containing active bacteria. The attempt itself is quite interesting. The reviewer will refer here on the side of the bacterium, G. xylinus as a component of the ink. In this ink, behavior of G. xylinus would be critical. The authors tried to control it by the oxygen supply, although the bacterium is quite sensitive to the environmental change (stress). In this point, there are several critical papers that the authors should need to cite; for example, Nagashima et al (2016) By considering the sensitivity of the bacterium to the culture condition and thereby feasibility of its changing behavior, the reviewer wonder if the obtained product could exhibit the stabilized quality. Therefore, the authors should be more careful in observing and realizing the bacterium behavior in the ink, not simply due to dependence on the oxygen supply.

Reviewer #1
Q1. The advantage of the newly introduced 3D-printing as described is not resolution and especially not complexity but the control over the topology and interconnectivity, which is a feature that is achieved uniquely by 3D-printing.
Response: We fully agree with the comment. The phrase "high resolutions" has been removed in the revised manuscript (MS). The MS has been modified to emphasize the features of SMAP as the reviewer mentioned. In addition, different designs for the 3D BC structures have been included in the revised MS. To clarify this point, the following text has been included: "The fabrication of a versatile free-form structure of bacterial cellulose (BC) has been proven impossible due to restricted oxygen supplies at the medium and the dimensional instability of hydrogel printing." "Complex 3D structures of BC with a resolution of about 50 µm were prepared in a mold with hydrophobic particles. Higher resolutions of the BC could be fabricated in patterned superhydrophobic-hydrophilic surfaces and at the interface of a soft-lithographic PDMS mold. Because of the limited choice of needles for direct 3D printing, the resolution of SMAP using a viscous ink is comparatively low. However, SMAP enables control over the topology and interconnectivity, which is a feature that is achieved uniquely by 3D printing." "Successfully constructed complex structures included coil, tetrahedron, connected ring, stacked lattice, and sandglass structures, which have not been reported in 3D printing to date (Fig. 1d)."

Q2. The disadvantages of 3D-printing (lower resolution) should be clearly highlighted as well as the advantages (higher versatility in topology).
Response: This point has been included in the revised MS. The following text has been included: "Complex 3D structures of BC with a resolution of about 50 µm were prepared in a mold with hydrophobic particles. Higher resolutions of the BC could be fabricated in patterned superhydrophobic-hydrophilic surfaces and at the interface of a soft-lithographic PDMS mold. Because of the limited choice of needles for direct 3D printing, the resolution of SMAP using a viscous ink is comparatively low. However, SMAP enables control over the topology and interconnectivity, which is a feature that is achieved uniquely by 3D printing." Response: We appreciate this comment. As the reviewer mentioned, SMAP is applicable in a variety of contexts. Here, we focused on bacterial cellulose, but SMAP can be expanded to the structures of other living inks. Furthermore, different types of solid particles, including metal, polymer, ceramic and wood, can be used as a solid matrix. Unfortunately, it is not possible to provide relevant experimental results, but this point has been included in the revised MS to inform the reader of the wide applicability of SMAP. To clarify this point, the following text has been included: "In contrast, SMAP allows for flexible design of more complex structures, including spheres, tubes, coils and connected rings. Especially, BC tubular structures can be used as engineered tissue support for blood vessels or neural regeneration. SMAP can be also applied to structure hydrogel inks containing various types of aerobic microorganisms. In addition, it is expected that SMAP will be expanded by diversifying the solid particles constituting the matrix such as metals, ceramics, and wood materials, which would upgrade the conventional 3D printing technology further." Response: We completely agree with the reviewer's comment. The statements have been corrected and modified in the revised MS. We focused on the higher versatility of the topology as a feature of SMAP in the revised MS. To clarify this point, the following text has been included:

Q4
"Complex 3D structures of BC with a resolution of about 50 µm were prepared in a mold with hydrophobic particles. Higher resolutions of the BC could be fabricated in patterned superhydrophobic-hydrophilic surfaces and at the interface of a soft-lithographic PDMS mold. Because of the limited choice of needles for direct 3D printing, the resolution of SMAP using a viscous ink is comparatively low. However, SMAP enables control over the topology and interconnectivity, which is a feature that is achieved uniquely by 3D printing."

Q5. A minor issue in Line 30
: "BC is a polysaccharide synthesized from Gluconacetobacter xylinus". Please note that many different microorganism strains also produce BC. IN passing, please note that authors refer to Acetobacter Xylinus in Fig. 1.
Response: We appreciate this comment. This error has been corrected in the revised MS.
"BC is a polysaccharide synthesized from many different microorganism strains and a hydrogel of a complex networked structure in which nanometer-sized cellulose fibers are intertwined." Reviewer #2: Q1. There is little information regarding the incubation time needed to produce these structures. Most samples were incubated for 7 days. In the case of the tubular structure, the thickness was evaluated and it is clear that after 8 days no more thickness can be achieved.
However, the blood vessel sample was incubated for 6 days. In static conditions, BC is often incubated for 7-14 days. Please, add some comments regarding why the authors used 7 days for most samples and if the incubation time could be controlled by modifying the glucose content of the ink.
Response: We appreciate the comments of the reviewer. In the case of the blood vessel sample, it was cultured for 7 days. This information has been corrected in the revised manuscript (MS).
Detailed information regarding BC production according to the incubation conditions, including glucose concentration, CNF concentration, and printing depth, have been included in the revised MS.
The following results are presented in Fig. 4 in the revised MS.
"For preparation of the blood vessel model, a hollow CNF/BC tube was prepared by printing a straight line of CNF hydrogel containing bacteria using SMAP, followed by incubation for 7 days and the subsequent removal of templating CNF hydrogel from the product." "The network structure of BC changes depending on the growing media conditions. The yield, fiber diameter, and mesh size of BC were analyzed by varying the concentration of mannitol, which was a carbon source contained in the ink, to 1.25, 2.5, 5, and 10%..........A straight line and the curved edge of BC structure produced at the same printing depth were characterized, and no significant differences in fiber diameter or mesh size of the BC were observed, implying the homogeneous BC hydrogels exhibited morphological diversity (Fig.   S2b, c)."

Q2. The authors should provide information regarding the properties of the BC network
(diameter and longitude of BC fibers, aspect ratio of BC fibers, mesh size of BC network).

The BC network properties depend on the growing media and conditions. For instance, the BC diameter should be compared with reported diameters of BC-based structures prepared with other techniques.
Response: We appreciate this comment. Unfortunately, it was not possible to provide the length or aspect ratio of the BC fibers because it is extremely difficult to take images of the full length of individual fibers. However, we analyzed the properties of the BC network, including the fiber diameter and mesh size, using FE-SEM images; this information has been included in the revised MS. The average diameters of BC fibers were found to be almost constant at about 55 nm. The mesh size of the BC was analyzed in accordance with the reference (Grande, Cristian J., et al., "Morphological characterization of bacterial cellulose-starch nanocomposites", Polymers and Polymer Composites 16.3 (2008): 181-185). In that paper, the mesh size of BC was defined as the distance between junction points. The diameters of the BC fibers appeared to be similar to those in previously reported studies. However, the mesh size varied according to the addition of CNF compared with the BC obtained using the static culture. We believe that the restricted cell locomotion in the CNF containing culture medium reduced the formation of BC. These results have been included in the revised MS.
Additionally, Fig. S2 has been included in the revised MS as follows: "The network structure of BC changes depending on the growing media conditions. The yield, fiber diameter, and mesh size of BC were analyzed by varying the concentration of mannitol, which was a carbon source contained in the ink, to 1.25, 2.5, 5, and 10%..........A straight line and the curved edge of BC structure produced at the same printing depth were characterized, and no significant differences in fiber diameter or mesh size of the BC were observed, implying the homogeneous BC hydrogels exhibited morphological diversity (Fig.   S2b, c)."

Q3. The authors should report if BC network properties (diameter, mesh size, etc.) are constant or depend on the structure they print.
Response: Thank you for your comment. We printed various structures and analyzed the network properties of BC using FE-SEM images. As shown in Fig. S2, it was confirmed that there were no significant changes in the fiber diameter or mesh size of BC. This is now described in the revised MS, and the following results are included in the Supporting information.
"Possible changes in the network structure of the BC according to the printed structure were investigated using two extreme shapes of BC (Fig. S2a). A straight line and the curved edge of BC structure produced at the same printing depth were characterized, and no significant differences in fiber diameter or mesh size of the BC were observed, implying the homogeneous BC hydrogels exhibited morphological diversity (Fig. S2b, c)." Q4. Regarding the rheological characterization of the BC-based hydrogels, the authors only presented strain sweeps. Why? Did the authors performed temperature and frequency sweeps?
Response: As the reviewer requested, we performed a frequency sweep to set the frequency for strain sweeping on the BC hydrogel. This information is included in the Supporting information. The change in shear modulus of the BC hydrogel was measured as the temperature was increased from 4 °C to 80 °C. The BC showed no significant change in shear modulus at all temperature conditions. This information is included in the Supporting information.
"Frequency sweep tests are widely used to obtain information regarding the stability of three-dimensional cross-linked networks. After the strain sweep test of Ca 2+ ions treated CNF/BC composites, the condition for the frequency sweeps was selected at 0.5% strain to ensure the linear viscoelastic range during the test (Fig. S5a). ……… G' and G'' values of Ca 2+ ions treated CNF/BC composites were constant in the temperature range from 4 °C to 80 °C confirming the high thermal stability of the hydrogel (Fig. S5b)."

Q5. Please report the thickness of the samples used for tensile tests.
Response: This information is included in the revised MS.

Please indicate the symbols used for the storage modulus (G´) and for the loss modulus (G´´).
Response: This is corrected in the revised MS.
Q7. Fig. 2e should be next (not below) to Fig. 2d Response: This is corrected in the revised MS.
Reviewer #3: Q1. The local amount of BC production in the hydrogels may significantly be influenced by the air-feeding levels. The even air-feeding is required to prepare homogeneous hydrogel thicknesses or amounts in throughout the hydrogel parts. How the authors controlled the homogeneous air-feeding in the whole interfaces?
Response: We agree with the reviewer's comment. As the reviewer mentioned, the supply of homogeneous air-feeding was one of the critical factors for the production of BC. The same volume of ink containing bacteria was printed in the solid matrix at a different printing depth. After incubation for 7 days, the weight, diameter and network density of the BC were measured. No significant differences in yield, diameter, or network density of the BC were observed, and it was determined that the solid matrix supplied even air-feeding in the printing environment.
"The production of BC in hydrogels may significantly be influenced by the air-feeding levels. It is critical to confirm even BC production with respect to the printing depth for the fabrication of homogeneous BC hydrogel structures. ……… Interestingly, the mesh sizes were also similar for different printing depths, which differs from the results obtained for different medium conditions (Fig. 4k, l)."

Q2. The information of the CM-CNF prepared in this study is insufficient. More detailed
information should be addressed in Supporting Information, such as the yield of CM-pulp, the degree of substitution, molecular weights, the degree of nanofibrillation, etc., otherwise readers cannot repeat the experiment. The details of kraft (not craft) pulp also should be addressed, such as the hemicellulose content, degree of polymerization, etc. In general, dissolving pulps with high cellulose contents are used in carboxymethylation. However, in this study, the kraft pulp containing a significant amount of hemicelluloses was used.
Please explain the reason.
Response: We agree with the reviewer's comments. More information regarding the CM-CNF and kraft pulp has been included in the revised MS. The kraft pulp was used to improve cost effectiveness and mass production in the future. To ensure reproducibility of the experiment, we agree that information on CM-CNF is important. The degree of substitution of CM-CNF was 1.14mmol/g and the yield was 80% based on the amount of CM-pulp obtained after the reaction. The degree of nanofibrillation was confirmed by AFM and the fiber diameter was measured to be 18.6 nm. The kraft pulp consisted of 79.4% ± 0.6% cellulose, 18.8% ± 0.2% hemicellulose and very small amounts of lignin and byproducts. The chemical composition of the pulp fiber was measured according to a TAPPI method (T 203 om-93). There was a paper that once-dried pulp with higher hemicellulose content that was fibrillated into 10−20 nm wide fibers as easily as the never- "The kraft pulp we used consists of 79.4% ± 0.6% cellulose, 18.8% ± 0.2% hemicellulose, and small amounts of lignin and byproducts." Q3. In this study, amidation was used to fix collagen to BC. Were the EDC, NHS used in this study are really safe to be used as artificial blood vessels? The amidation used in this system is not simple, and some residual EDC or NHS may remain in the hydrogels as counterions of carboxy groups of CM-CNF.
Response: We appreciate the comment of the reviewer. We used EDC/NHS chemistry to conjugate collagen to the BC/CM-CNF complex. Bioconjugation using EDC and NHS has been admitted generally as a non-cytotoxic and biocompatible chemistry. Response: We agree with this comment. We further investigated the stabilized quality of BC production. The yield, fiber diameter, and network density of BC were characterized according to the medium conditions, including the concentrations of glucose and CNF, as well as the printing depth of the solid matrix and printing structures. This information has been included in the revised MS.
"The network structure of BC changes depending on the growing media conditions. The yield, fiber diameter, and mesh size of BC were analyzed by varying the concentration of mannitol, which was a carbon source contained in the ink, to 1.25, 2.5, 5, and 10%..........A straight line and the curved edge of BC structure produced at the same printing depth were characterized, and no significant differences in fiber diameter or mesh size of the BC were