RESEARCH ARTICLE www.advancedscience.com Highly Aligned Bacterial Nanocellulose Films Obtained During Static Biosynthesis in a Reproducible and Straightforward Approach Nerea Murugarren, Soledad Roig-Sanchez, Irene Antón-Sales, Nanthilde Malandain, Kai Xu, Eduardo Solano, Juan Sebastian Reparaz, and Anna Laromaine* 1. Introduction Bacterial nanocellulose (BNC) is usually produced as randomly-organized highly pure cellulose nanofibers films. Its high water-holding capacity, Anisotropic structures are ubiquitous in na- ture and our tissues, and we find them porosity, mechanical strength, and biocompatibility make it unique. Ordered carrying out particular functions, includ- structures are found in nature and the properties appearing upon aligning ing mass transport, surface lubrication, cell polymers fibers inspire everyone to achieve highly aligned BNC (A-BNC) films. adhesion, and force generation. For in- This work takes advantage of natural bacteria biosynthesis in a reproducible stance, aligned actin fibers confer strong and straightforward approach. Bacteria confined and statically incubated muscle resistance, and highly aligned col- biosynthesized BNC nanofibers in a single direction without entanglement. lagen fibers offer a transparent stromal tissue[1,2] or strengthen the skin[3] and ar- The obtained film is highly oriented within the total volume confirmed by ticular cartilages.[4] Additionally, the align- polarization-resolved second-harmonic generation signal and Small Angle ment of fibers usually confers novel and ex- X-ray Scattering. The biosynthesis approach is improved by reusing the citing properties to polymers, such as piezo- bacterial substrates to obtain A-BNC reproducibly and repeatedly. The electricity, conductivity, or light-harnessing suitability of A-BNC as cell carriers is confirmed by adhering to and growing properties.[5–9] Cellulose is the most abundant biopoly- fibroblasts in the substrate. Finally, the thermal conductivity is evaluated by mer in nature,[10] and it is commonly two independent approaches, i.e., using the well-known 3𝝎-method and a obtained from plants, even though sev- recently developed contactless thermoreflectance approach, confirming a eral organisms also present the ability to thermal conductivity of 1.63 W mK−1 in the direction of the aligned fibers produce it, such as algae,[11] fungi, and versus 0.3 W mK−1 perpendicularly. The fivefold increase in thermal bacteria.[12] The nonpathogenic bacteria Ko- conductivity of BNC in the alignment direction forecasts the potential of magataeibacter xylinus (K. xylinus) extrudes bacterial nanocellulose (BNC) fibers at the BNC-based devices outperforming some other natural polymer and synthetic liquid–air interface.[13,14] BNC is a semicrys- materials. talline, hierarchical, and fibrillar network that confers properties such as high water holding capacity, porosity, mechanically solid, and elastic, mak- ing the biomaterial highly biocompatible and with bio-welding properties.[14–22] Those extraordinary properties placed BNC at the intersection of different applications. N. Murugarren, S. Roig-Sanchez, I. Antón-Sales, N. Malandain, K. Xu, BNC field advances to control the intrinsic properties of cel- J. S. Reparaz, A. Laromaine Institut Ciencia de Materials de Barcelona (ICMAB-CSIC) lulose fibrils, such as their arrangement, their interactions with Campus UAB, Bellaterra 08193, Spain proteins and solvents, or the addition of additives to endow E-mail: alaromaine@icmab.es novel properties to the material, among others.[23–25] Under- E. Solano standing the basic material and the novel composites have en- NCD-SWEET beamline dorsed its uses in fields ranging from batteries[26–28] up to skin ALBA Synchrotron Light Source Carrer de la Llum 2−26, Cerdanyola del Vallès, Barcelona 08290, Spain grafts,[17–19,29,30] or even heart replacement pouches.[31–33] Cellulose and bacterial nanocellulose fibers are promising building blocks for high-performance biomaterials, especially The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/advs.202201947 if we can control their intrinsic mesh structure. BNC fibers are long, flexible, and highly entangled, as obtained; therefore, © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons controlling the alignment of nanofibers is challenging. As re- Attribution License, which permits use, distribution and reproduction in viewed by Li et al.[34] different approaches have been presented any medium, provided the original work is properly cited. since bacterial cellulose fibers’ alignment could have interesting DOI: 10.1002/advs.202201947 properties. Top-down and bottom-up approaches have been Adv. Sci. 2022, 9, 2201947 2201947 (1 of 14) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH www.advancedsciencenews.com www.advancedscience.com Figure 1. Methodologies to align BNC fibers. A,B) Schematics of the reported methods to align bacterial cellulose fibers such as top-down approaches: uniaxial stretching,[35–37] wet spinning,[38] and wet-drawing and wet-twisting[39] to name some and bottom-up such as flow-induced alignment in bioreactors,[40] patterned templates,[42,43] physical and oxygen gradients.[42,43] Bottom-up methodologies benefit from the use of BNC biosynthesis. C) Illustration of the natural BNC biosynthesis and the structural hierarchy of BNC fibers. K. xylinus, a cellulose-producing bacteria strain, synthesizes cellulose from the glucose present in the culture media through the enzymes GK (glucokinase), PGM (phosphoglucomutase), UGP (UDPGlc pyrophos- phorylase), and CS (cellulose synthase).[43] Cellulose is first produced as elementary fibrils, a cellulose chain of glucose units bound by 𝛽-1,4 glycosidic bonds then assembled into microfibrils, which are in turn assembled into microfibrils bundles. The last assembly is the cellulose fiber, which in the standard biosynthesis process in static forms a BNC pellicle at the liquid–air interface. The illustration scales are not representative of reality. D = diameter. reported, as illustrated in Figure 1A,B. Top-down methods, such systems to produce and control oxygen and physical gradients in as an uniaxial stretching of BNC wet hydrogels,[35–37] the use patterned and/or modified surfaces. of wet spinning[38] of a BNC solution, and wet-drawing and Due to the difficulty of maintaining the aligned structures wet-twisting of BNC films[39] produced strong, long, and tough stable,[34,41] polymers (PVA or PLGA), ionic crosslinkers[38] or aligned BNC films. Yao et al.[38] obtained highly aligned BNC, other additives are often required to improve and stabilize the which they evaluated with the orientation parameters[34] Orien- aligned BNC films. For example, Rahman et al.[36] added soy pro- tation Index (OI) and Herman’s order parameter (S-parameter) tein to uniaxial-stretched BNC films to enhance the mechanical (Figure 1B), parameters widely accepted to describe anisotropic properties by 2.75 and threefold in tensile strength and tensile fibrillar materials.[34] However, to keep the alignment structure modulus, respectively. stable, TEMPO (2,2,6,6-Tetramethylpiperidinyloxy, a mediator in Even though there are some examples of materials contain- radical chemistry) oxidation processes, wet spinning of the BNC ing aligned BNC fibers, yet the methods to produce them are not fibers solutions, stretching and ionic crosslinking of the BNC straightforward. We designed a methodology to obtain aligned substrate were required. On the other hand, bottom-up strate- BNC films in static conditions and avoid complex equipment or gies produced oriented materials by bacteria biosynthesis. Luo chemical additives. For that, we observed the BNC synthesis. K. et al.[40] obtained oriented BNC fibers by forcing the movement xylinus produces cellulose, as described in Figure 1C, involving a of the culture in a single direction in a bioreactor. While an inter- chain of biochemical reactions. Cellulose fibers are extruded lin- esting approach, this methodology produced bacteria mutations, early through the bacteria cell wall’s pores,[6,14,44,45] as recorded by decreasing the production yield.[40,41] Significantly, Putra et al.[42] R. Malcolm Brown, JR. laboratory.[45] Cellulose ensembles in hi- and Greca et al.[43] also benefitted from the bacterial culture erarchical structures from elementary fibrils to BNC fibers[21,22] and obtained aligned BNC using interesting, although complex, (terminology used in the text), and forms a final BNC pellicle at Adv. Sci. 2022, 9, 2201947 2201947 (2 of 14) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH www.advancedsciencenews.com www.advancedscience.com Figure 2. Methodology to obtain aligned BNC (A-BNC) and the reuse of the bacterial substrates. A) Screenshot of the extrusion of BNC fibrils by K. xylinus strain’s individual pores when immersed in HS liquid culture media with no restriction of movement. B) K. xylinus colonies grow confluent on the surface of HS solid media (agar). C) Square section of agar with confluent bacterial colonies (marked in blue) is placed at the bottom of the culture tube filled with HS liquid culture media, restricting the bacteria movement. Due to the oxygen availability, bacteria move toward the liquid–air interface to reach higher DO concentrations, leaving aligned BNC (A-BNC) fibers behind. Hence, A-BNC is synthesized with directionality toward the liquid–air interface of the culture, forming BNC. D,E) Optical images of the macrostructure of A-BNC within the length of a A-BNC and BNC film. F) Reuse of bacterial substrates: agar containing bacterial colonies (marked as blue) and the produced BNC film (marked as green) are reused to grow aligned BNC. G) Reusing the agar square piece, which still maintains bacterial confluence, at the bottom of the vessel of a new culture tube allows obtaining A-BNC and BNC repeatedly for at least 7 cycles. H) Reusing the produced and not cleaned BNC film (marked in green in F) allowed the “Film-to-film” biosynthesis method. I) Scanning electron microscopy (SEM) image of an uncleaned BNC film containing bacteria. J) Reusing uncleaned BNC films in a new culture tube allowed obtaining “Film-to-film,” which consist in 2 BNC films bound at the extremes of an A-BNC film. This process can be repeated multiple times and avoiding bacteria and time waste. the liquid–air interface in static biosynthesis. Motivated by this Figure S1, Supporting Information). As previously reported, we biosynthesis process, we investigated whether the restriction of obtained the typical hydrogel pellicle of BNC at the liquid-air in- the bacteria’s movement could retain the intrinsic parallel orien- terface after 5 days of culture[14,44] (Figure 2C). The appearance of tation of individual cellulose fibers.[44] Here, we report a novel, this film indicated that some bacteria reached the surface from reproducible, and easy biofabrication method to obtain aligned the vessel’s bottom and were able to produce cellulosic fibers at BNC (A-BNC) hydrogels with high control, avoiding complex the liquid–air interface. As a result of the migration from the ves- equipment and additives. The morphology, optical activity, and sel’s bottom to the surface, a translucent film of aligned BNC how the anisotropy of this material impacts its hydrophilicity, bio- fibers is formed in the liquid spanning the whole length of the compatibility, and thermal conductivity of A-BNC films are eval- culture vessel after a biosynthesis cycle, as shown in Figure 2D,F. uated. This methodology was repeated n ≥ 200, obtaining freestanding A-BNC and BNC films consistently (Figure S2A, Supporting In- 2. Results and Discussion formation). From each culture, we isolated the aligned cellulose (A-BNC), which was bound to the agar substrate from the bottom 2.1. Aligned Bacterial Cellulose Biosynthesis of the vessel and to the cellulose hydrogel pellicles obtained at the top of the vessel (BNC) (Figure 2D). The restriction of the bac- K. xylinus is an aerobic bacteria strain[6,44] and typically produces teria movement allowed to produce fibers without the entangle- BNC films which at harvested at the liquid–air interface in the ment in a standard bacterial liquid culture. Hence, we obtained culture. Bacteria in solution can move without any restriction of BNC films of different morphologies (aligned and nonaligned) movement Figures 1C and 2A and drives the production of the in a straightforward step. water-insoluble[14] cellulose nanofibrils toward a higher oxygen We cleaned the films from bacteria detritus and culture me- content.[42,43] In our approach, we first cultured confluent K. xyli- dia using the standard treatment described previously.[46] We ob- nus colonies on an agar surface containing nutrients for 15 days at tained semitransparent hydrogels of BNC of the vessel’s shape, 30 °C (Figure 2B). Then, we cut square pieces of the agar with the in our case disks of Ø1.6 cm,[2] and aligned bacterial nanocellu- colonies and place those agar squares with the bacterial colonies lose (A-BNC) of 7–8 cm in length. A-BNC and BNC films were facing up at the bottom of a vessel, previously filled with fresh dried at 60 °C for a minimum of 12 h. After drying, A-BNC bacteria-free liquid media. Bacteria anchored at the agar started maintained the aligned structure and length observed in the wet to produce cellulose at 30 °C in static conditions (Figure 2C; and state. A-BNC films were easily manipulable and flexible, with a Adv. Sci. 2022, 9, 2201947 2201947 (3 of 14) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH www.advancedsciencenews.com www.advancedscience.com thickness of ≈10 μm after drying. Optical images of the A-BNC ure 2I). As shown in Figure 2H, uncleaned BNC films (marked clearly show the alignment of the fibers (Figure 2C,D); con- in green) containing live and active bacteria allow producing A- versely, the random BNC does not show any orientation of its BNC films resembling agar substrates. BNC films could easily be structure. detached from A-BNC by pulling off the film with Teflon tweezers Interestingly, A-BNC did not disassemble upon different and cutting with sterilized scissors. When placed at the bottom of wetting-drying cycles and the whole cleaning process, indicat- a vessel, the bacteria immediately produce cellulose fibers. This ing a stable cellulose fibers interaction conferred by hydrogen results in films containing 2 circular BNC pellicles bound at the bonds between hydroxyl groups.[10] This is a key feature as re- extremes of an A-BNC film (a “Film-to-film”, Figure 2J), which ported aligned films usually require additives and crosslinkers to allows the production (2n ) of A-BNC films. This process could be maintain the structure.[34,38] used repeated for at least 7 cycles, obtaining freestanding “Film- Many parameters control the BNC production, and they have to-film” consistently (Figure S2B, Supporting Information). The been evaluated elsewhere.[14,43,46] However, the media’s dissolved re-use of agar and uncleaned BNC film substrates in the A-BNC oxygen (DO) concentration is considered the main driver (Fig- production allowed to increase exponentially the number of films ure 2C). We quantified the DO concentration present in the so- obtained. We hypothesize that the already-used bacteria are accli- lution at different time points using the Leuco-indigo carmine matized to the liquid culture, reducing the lag phase of a bacteria reaction[47,48] (Figure S3, Supporting Information) in our system. culture from the initial 5 days of the first culture to only 3 days, Leuco-indigo carmine reagent oxidizes under the presence of DO speeding the biosynthesis process.[41,49] to keto-indigo carmine, which in turn causes a color change in the To increase the dimensions of the A-BNC produced, we eval- solution from yellow to red upon oxidation (Figure S3A, Support- uate the culture time, vessel dimensions, and bacteria amount. ing Information). A color chart allows to qualitatively estimate Culturing the bacteria from 3 to 15 days did not increase the the DO concentration (Figure S3B, Supporting Information). As width or length of A-BNC, although it increased the thickness shown in Figure S3C (Supporting Information), the initial DO of the BNC film. On the other hand, longer A-BNC films could concentration at the top, and bottom part of a nonoxygen perme- be obtained using longer culture tubes. Using tubes of 10 and able tube was ≈0.06 ppm. After 7 days of culture, the DO con- 30 cm allowed obtaining A-BNC 7–8 cm up to 17–20 cm, respec- centration at the top was maintained at 0.06 ppm while, at the tively; however, the width of the A-BNC was not increased (3– bottom, it decreased to 0.03 ppm. We hypothesize that the deploy- 5 mm). Figure S5 shows images of the methodology to obtain ment of oxygen at the bottom of the vessel over time promotes the A-BNC films in large vessels (30 cm). Finally, instead of using movement of the bacteria to the surface toward higher DO con- the typical streak plate (or zig–zag) method to obtain the initial tent. Thus, bacteria produce water-insoluble cellulose fibers from agar substrate with bacterial colonies,[50] we grew confluent agar the bottom of the vessel and use them as “climbing ropes” (Fig- Petri dishes to maximize the number of bacteria per surface to ure 2C) to reach the high oxygen-content surface where they con- obtain high-density A-BNC fiber films. This strategy allowed us tinue producing its exoproduct with no movement restriction, to obtain A-BNC films with a thickness of ≈200 μm in wet. allowing us to obtain BNC as a random mesh. Remarkably, we efficiently obtain A-BNC and BNC from the same biosynthesis process by restricting the initial movement of bacteria and its en- 2.2. A-BNC Characterization tanglement. To further exploit the efficiency and yield of our new method, We extensively characterized A-BNC and BNC (considered a con- we investigated the reuse of the agar containing bacterial colonies trol) to evidence the change of morphology and evaluated the and the produced and uncleaned BNC films (marked in blue and properties arising upon the alignment of the fibers. green in Figure 2F, respectively). After an A-BNC and BNC film After drying, A-BNC is more transparent than BNC, qualita- culture, the agar substrates did not lose the confluence of bacte- tively appreciated by the naked eye (Figure 3A). Figure 3B,C show rial colonies, which confirmed its suitability for our methodology pictures taken with a micrometer-mesh pattern under the A-BNC based in the restriction of the bacteria movement. In Figure S4 and BNC films, respectively, and top illumination. The scatter- (Supporting Information), bacterial colonies can be observed at ing of light of BNC produced a haze and whitish color to the the extreme of an A-BNC and BNC film, which was pulled from material that was not seen in the A-BNC. Moreover, under the the agar substrate. This piece of agar was carefully released from incidence of white light, the A-BNC films showed iridescence, the produced cellulosic film and confined again at the bottom of indicating higher transparency and structuration of the material a new culture tube previously filled with fresh liquid culture me- (Figure 3D). dia (free of bacteria) (Figure 2G). We were able to reuse the same As seen in Figure 3E, FTIR for both samples (BNC (blue) agar substrates with confluent bacterial colonies (Figure 2G) for and A-BNC (black)) contained the cellulose characteristics peaks: at least 7 cycles, obtaining A-BNC and BNC films in only 3 days. hydroxyl groups are seen as a strong peak at 3000–3700 cm−1 , After that, the bacteria decreased the speed of production. K. xyli- CH groups at 2800–3100 cm−1 , and the internal deformation nus exhibits the growth dynamics of a common bacteria, decreas- frequencies of CH2 and CH3 groups appeared in the region of ing its activity and efficiency to produce BNC over time since it 1340–1480 cm−1 . However, the A-BNC spectra show more in- enters the death phase, where there is less metabolic activity.[41,49] tense transmittance peaks in the OH and CH region, characteris- We also explored the reuse of the uncleaned BNC film produced tic of cellulose, even though A-BNC films were thinner than the at the liquid–air interface, which we named “Film-to-film” syn- BNC. thesis (Figure 2H). By scanning electron microscopy (SEM) we To analyze the BNC film at the nanoscale (Figure 4), films were detected the presence of bacteria in uncleaned BNC films (Fig- dried, which collapsed the structure and made the alignment Adv. Sci. 2022, 9, 2201947 2201947 (4 of 14) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH www.advancedsciencenews.com www.advancedscience.com puted the local orientation as a color-survey HSB image, where hue represents the fiber orientation, and saturation represents orientation coherency. These images (insets of Figure 4C,F) indicated the preferential fiber orientation in degrees. We can see that A-BNC had a predominance of colors in the green (Figure 4F) region, indicating a predominant direction (pointed by white arrows for a more precise visualization) not appreciable for BNC samples (Figure 4C), where a variety of colors represent the different orientations within the image. The mean height of AFM images analyzed by MountainView8 was 28.6 nm for BNC and 21.1 nm for A-BNC, indicating a flatter surface. Additionally, we computed the isotropy of the pictures in Figure 4D,G, show- ing higher values for BNC, 31%, in contrast with A-BNC, 19%, confirming the alignment. Interferometry assisted us in visualiz- ing the alignment at a micron scale, obtaining volumetric images of the aligned cellulose BNC bundles (Figure S7A, Supporting Information), in contrast with the nonaligned BNC (Fig- ure S7B, Supporting Information), where nonbundle ordering is appreciated. To evaluate the alignment in the 3D volume of the mate- rial, we used interferometry, small angle x-ray scattering (SAXS), and polarization-resolved second-harmonic generation signal (P- SHG). The raw 2D scattering SAXS patterns of A-BNC and BNC fibers showed a different scattering pattern shape, being circu- lar for randomly distributed fibers of the BNC (Figure 5A) and distorted (elliptical) for preferential orientated fibers of the A- BNC sample (Figure 5B), indicating a high degree of alignment. The spectra were integrated to quantitatively evaluate differences in the orientation degree, as shown in Figure 5C, as of inten- Figure 3. Transparency and chemistry of A-BNC. A) Pictures of A-BNC and BNC, respectively. B,C) Pictures of A-BNC and BNC on top of a sity versus azimuthal degreeplot from −90° to 90°. A-BNC ex- micrometer-mesh, indicating a hazier BNC than A-BNC. D) Dry A-BNC hibited an intensity peak centered around 0°, whereas BNC gave shows iridescence effects under the incidence of white light. E) FTIR re- no peak. From the widely accepted orientation quantitative pa- veals that A-BNC and BNC have similar chemical compositions. However, rameters orientation index (OI), full width at the half-maximum the A-BNC spectra show more intense transmittance peaks in the OH and intensity (FWHM), and S-parameter[34,38] obtained from the spec- CH region. tra where FWHM stands for full width at the half-maximum in- tensity and the smaller this value is, the highest is the orienta- tion. The OI is obtained from FWHM, ranging from 0 for ran- observed at the macroscale more difficult. Even though a higher domly oriented structures to 1 for perfectly aligned fibers. Her- degree of organization was observed in the A-BNC than in BNC. man’s order parameter, also called S-parameter, can be calcu- Figure 4B,E shows the transmission electron microscopy (TEM) lated from the intensity and azimuthal degrees values, and it images and selected area electron diffraction (SAED) patterns ranges between 0 and 1, being 1 a perfect oriented.[31,34] Thus, of BNC (Figure 4B) and A-BNC (Figure 4E). Although BNC is as shown in Figure 5D, FWHM, OI, and S-parameter were com- semicrystalline (60–80%),[44] the thickness of the film, compact- puted, obtaining 47.18°, 0.74, and 0.85 for A-BNC, respectively. ness, and orientation of the fibers make it challenging to analyze For BNC, S-parameter resulted to be much smaller than A-BNC, the crystallinity of the BNC pellicle by TEM.[51] SAED analysis 0.16. FWHM and OI could not be determined due to the no peak in A-BNC samples allowed visualizing the cellulose crystallinity presence in the spectra, thus lacking internal orientation. The nu- lattice planes (indexed in Figure S6A, Supporting Information). merical results obtained corroborated the qualitative information We considered that this increased discretization of the lattice extracted from the 2D images. The orientation values OI and S- planes intensity (from diffuse bands to localized bright spots, Fig- parameter (0.74 and 0.85, respectively) were higher than previ- ure S6, Supporting Information) was due to the increased order ously reported values of aligned BNC fibers obtained by wet spin- orientation and different textures of A-BNC. Scanning electron ning, with OI of 0.69 and an S-parameter of 0.63.[31] microscopy (SEM) and atomic force microscopy (AFM) images Secondary harmonic generation (SHG) is a nonlinear optical show a similar compact structure for the BNC (Figure 4C,D) and process used in biomedicine for imaging SHG active biological A-BNC (Figure 4F,G) films; however, we could see the alignment structures such as collagen nanofibers, rather than bulk.[43,52,54] of fibers in A-BNC. Diameter measurements of the nanofibers Polarization-resolved SHG microscopy (PSHG) is a powerful tool (2–4 nm) and microfibrils bundles (15–20 nm) from both A-BNC that exploits the dependence of SHG signals on the polarization and BNC revealed that the modification in the culture to obtain degree of the excitation beam.[52] The SHG emission depends on aligned BNC did not impact the single fiber structure. Using the the structuration of the imaged material and PSH includes an ex- color analysis from the OrientationJ plugin for ImageJ, we com- tra dimension that is used to probe the molecular organization. Adv. Sci. 2022, 9, 2201947 2201947 (5 of 14) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH www.advancedsciencenews.com www.advancedscience.com Figure 4. Morphology and structure analysis. A) Picture of the as-produced nanocellulose films containing BNC and A-BNC. The top B,C,D) and bottom E,F,G) rows contain the images obtained by TEM, SEM, and AFM of BNC and A-BNC. B,E) TEM images and selected area electron diffraction (SAED) patterns of BNC and A-BNC samples. C,F) SEM micrographs of BNC and A-BNC samples (Insets: color analysis from the OrientationJ plugin for ImageJ). D,G) AFM images of BNC and A-BNC samples (Insets: isotropy distribution computed using MountainView8). Figure 5. Nanoscale density study with SAXS. A,B) Small angle X-ray scattering (SAXS) scattering signal of BNC and A-BNC. C) Integration of the spectra as intensity versus azimuthal degree plot, from −90° to 90°, whereas BNC gave no peak. D) Table with the quantitative orientation parameters orientation index (OI), full width at the half-maximum intensity (FWHM), and Herman’s order parameter (S-parameter) values for BNC and A-BNC. Adv. Sci. 2022, 9, 2201947 2201947 (6 of 14) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH www.advancedsciencenews.com www.advancedscience.com Figure 6. Polarization-resolved second harmonic generation (PSHG) microscopy. Stacks of 18 images (frame/10° light polarization) were analyzed with ImageJ using a heat map and Z projections. A) Maximum and minimum SHG intensity projection of 150 × 150 μm2 for BNC and A-BNC, along with the intensity color map. B) Plot of the SHG intensity for each angle polarization, along with their alignment coherency, obtained with the OrientationJ plugin for ImageJ. Here, PSHG was used to assess the bacterial nanocellulose in- Figure 7C,F, N-shaped films of A-BNC created a pattern of light trinsic organization. Frames of the linear polarization of the inci- polarization at different positions. dent beam from 0° to 180° in steps of 10° using a 𝜆/2 plate were recorded for the whole volume of the samples. Figure 6A shows the changes of light intensity in A-BNC from the minimal (70°) to 2.4. Hydrophilicity and Cell Substrates Evaluation the maximum projection (150°), whereas, in BNC, all projections remained unchanged, indicating no alignment. These features Most of the electrospun polymers are relatively hydrophobic, were observed for the whole thickness of the samples, ensuring which is unfavorable from the point of view of tissue engineering. a volumetric alignment in A-BNC. We evaluated the alignment For instance, aliphatic polyesters such as Poly(L-lactide) (PLLA) coherency for each frame by the OrientationJ plugin for ImageJ or Polycaprolactone (PCL) show contact angles in the range (Figure 6B). Random BNC displays a high but invariable signal of 116°–135°, while tissue engineering requirements ought to intensity (blue line) and a low alignment coherency (dotted blue be below 100°.[55,56] Webb et al. and Niemczyk et al. studied line). Conversely, A-BNC alignment coherency (dotted black line) the highest level of fibroblasts cell attachment at hydrophilic is even higher than the signal intensity (black line) at the maxi- surfaces,[57,58] and the best results were observed for surfaces mum projection and lower in the minimum projection, confirm- with contact angles in the range of 20°–40°. As cellulose, and ing the alignment of the material. specifically, bacterial nanocellulose drives many applications in medicine[59,60] for their biocompatibility and hydrophilic charac- ter, we wanted to analyze the impact of the fiber alignment in 2.3. Optical Properties the hydrophilic nature of bacterial cellulose. Some reports al- ready suggest the importance of structuration of cellulose on the As we have presented, A-BNC films exhibit iridescence and growth of specific cell lines.[33,61] We deposited a blue-colored strong light polarization properties. The use of polarized light is drop of water over A-BNC film, and we could see a faster hori- a well-known methodology to assess a material’s anisotropy.[40,42] zontally spreading of the liquid than BNC, indicating a different As shown in Figure 7, a dark-light patterns can be obtained when structure (Figure 8A). To quantify the hydrophilicity of both A- a sample is circularly rotated 45°. The minimum light intensity BNC and BNC, we performed “apparent contact angle” (ACA) is obtained when the fibers are placed at 0° from the incident po- measurements. After 5 s of depositing a water drop over the sur- larized light, whereas the maximum light intensity of the fibers faces, we observed an ACA of 0° for A-BNC (Figure 8B), whereas appear at 45°. BNC films did not exhibit light polarization at 0° it remained 29.7° for BNC after 5 s (Figure 8C). These results and 45° (Figure 7A,D), confirming the random distribution of the indicated that the alignment of fibers seemed to increase the fibers. On the other hand, A-BNC films gave dark-light patterns hydrophilicity of the films, which also matches with the lower of light when disposed at 0° and 45° from the incident polarized roughness of A-BNC in AFM and the increased density of OH light beam, respectively (Figure 7B,E), confirming the anisotropy groups by FTIR. of the material. Moreover, A-BNC is a flexible material and can This change of hydrophilicity was further explored in vitro be folded into any desirable shape, such as an “N”. As seen in in cell cultures. A human dermal fibroblast (hDF) cell line Adv. Sci. 2022, 9, 2201947 2201947 (7 of 14) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH www.advancedsciencenews.com www.advancedscience.com Figure 7. The behavior of A-BNC and BNC under polarized light. Visualization under a polarized light microscope at 0° and 45° (incident polarized light beam vs sample orientation) of A,D) BNC samples, B,E) A-BNC samples and C,F) N-shaped A-BNC (Insets of whole films in (A)–(C)). Samples are circularly rotated within the same plane, perpendicularly to the incident polarized light beam. Figure 8. Hydrophilicity and cell substrates evaluation of A-BNC and BNC. A) Pictures of an A-BNC film’s wetting with a colored water drop. B,C) Measurement of the apparent contact angle (ACA) of A-BNC and BNC films, respectively. D,E) Optical images of confluent cell cultures at day 7 on A-BNC and BNC, respectively. F) Orientation coherency plot of A-BNC, BNC, and the slide (control), computed with the OrientationJ plugin of ImageJ. *** = P ≤ 0.001, **** = P ≤ 0.0001. Adv. Sci. 2022, 9, 2201947 2201947 (8 of 14) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH www.advancedsciencenews.com www.advancedscience.com Figure 9. Thermal conductivity study of A-BNC. A) 3𝜔 measurements of A-BNC at the parallel and perpendicular directions were evaluated, indicating a maximum five-fold increase of the thermal conductivity in the parallel direction of A-BNC fibers. B) Thermal conductivity of A-BNC measured by anisotropic thermoreflectance thermometry, showing similar results as shown in (A). C) Comparison of the obtained measurements to the standard collagen,[65] PLA,[70] or PE[68] used in implants. D) Table of the reported thermal conductivity of polymeric materials.[62,65,68,70–73] (1BR.3.G) was cultured on A-BNC and BNC films. The cell at- fibers, i.e., in the direction parallel to the alignment direction. tachment and growth directionality were assessed visually and We used two independent approaches to investigate the thermal with orientation coherency measurements with the OrientationJ conductivity of A-BNC, using the well-known 3𝜔-method,[65] and plugin of ImageJ. As seen in Figure S8 (Supporting Information), a recently developed contactless thermoreflectance frequency- cells attached and proliferated on A-BNC, BNC, and control (glass domain method developed by Pérez et al.,[66] named anisotropic slide) until reaching a 100% confluence at day 7 (Figure S8, Sup- thermoreflectance thermometry (ATT). The latter methodology porting Information; and Figure 8D,E). As seen in Figure 8F, is particularly suitable for studying thermally anisotropic mate- hDF cells cultured on aligned cellulose fibers tend to be more rials (e.g., aligned BNC), since it delivers the angular distribu- elongated (orientation coherency of 0.25) than on random fiber tion of the thermal conductivity perpendicular to the sample’s distributions (BNC film) (orientation coherency of 0.12) and on surface. In all cases, the BNC fibers were deposited on silicon the slide (orientation coherency of 0.8) The increase of hDF direc- substrates, and the thickness of the BNC samples was ≈10 μm. tionality indicated that cells perceived at some extend the align- Figure 9A displays the 3𝜔 measurements in perpendicular (red) ment of the cellulosic fibers. and parallel (blue) to the alignment direction. Whereas the lower frequency range is dominated by the thermal response of the Si substrate, the higher frequency range contains information on 2.5. A-BNC Thermal Conductivity the thermal properties of the fibers. The thermal conductivity is estimated using the “slope method” (𝜅 𝛼 [∂V3𝜔 /∂ log( f )]−1 ) in The advent of more sophisticated implants combining novel bio- the higher frequency range. The lower frequency range is dom- materials, polymers, electrical components, among others, urges inated by a thermal signal from the substrate, and it is used as precise thermal management.[62] Amorphous materials and good calibration for each measurement (𝜅 Si = 150 W m−1 K−1 ). In the thermal insulators tend to be abundant in biologically derived parallel direction, A-BNC shows a more significant thermal con- materials; to enhance their thermal conductivity, self-assembled ductivity at 1.63 ± 0.15 W m−1 K−1 , whereas in the perpendicu- biomaterials with a higher degree of crystallinity and higher lar direction, the thermal conductivity is 0.3 ± 0.02 W m−1 K−1 , anisotropy are desirable.[63] BNC has already shown promising slightly lower than what we previously obtained for BNC, 0.5 ± results as a scaffold in regenerative medicine,[17-19,64] we studied 0.05 W m−1 K−1 .[7] The alignment of the fibers led to a fivefold the influence of the geometrical order on the thermal conduc- increase of the thermal conductivity of BNC in the parallel direc- tivity of BNC fibers. In particular, we show that it is possible to tion (Figure 9A). Similar results were obtained with ATT, where enhance the thermal conductivity by directional alignment of the thermal conductivity was measured almost continuously with 1° Adv. Sci. 2022, 9, 2201947 2201947 (9 of 14) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH www.advancedsciencenews.com www.advancedscience.com angular steps from the alignment direction (Figure 9B). Those (NCIMB 5346 from CECT, Valencia, Spain) were grown confluent on results indicate that the heat transport is more efficient in the Hestrin–Schramm (HS) solid media[13] in Petri dishes. The HS solid me- parallel direction rather than the perpendicular. The main lim- dia consists of 1.15 g L−1 of citric acid, 6.80 g L−1 of Na2 HPO4 ·12H2O, 5.00 g L−1 of peptone, 5.00 g L−1 of yeast extract, 15.00 g L−1 of agar, itation in the perpendicular direction is the dominant thermal and 20.00 g L−1 of dextrose all from Condalab, dissolved and autoclaved interface resistance between fibers since these are probably in- in 1 L Milli-Q water. HS liquid media contained the same components teracting. This increase in heat transport in aligned polymers has as the HS solid media but without agar (jelling agent). K. xylinus strain been already reported, for instance for polyethylene.[67] The high- was cultured in HS solid media Petri plates for 15 days at 30 °C. Next, est value for A-BNC, at its parallel direction, (1.42 ± 0.15 W m−1 agar squares of ≈70 mm side with K. xylinus grown colonies were cut and K−1 ), is similar to the value obtained using the 3𝜔-method (1.63 ± placed carefully facing up with a sterile spatula at the bottom of the cul- ture tube, previously filled with 10 mL sterile bacteria-free liquid HS media 0.15 W m−1 K−1 ), This number is on the same order as polyethy- with an oxygen-permeable cap. Due to the oxygen availability that an aero- lene (PE) (1.33 W m−1 K−1 ),[68] and larger than collagen (0.53 W bic bacteria strain (K. xylinus) require to survive, the dissolved oxygen (DO) m−1 K−1 ),[65] which are materials commonly used in implants. concentration gradient formed in the culture tube is the main driving force Therefore, the geometrical alignment of the fibers offers the pos- of this method. The bacteria move toward the liquid–air interface to reach sibility to control at different planes the thermal conductivity just higher DO concentrations, leaving aligned BNC (A-BNC) fibers behind. by tailoring the anisotropy of the biomaterial.[69] Interestingly, the Hence, A-BNC is synthesized with directionality toward the liquid–air in- perpendicular component of the thermal conductivity from the terface of the culture. Once A-BNC synthesis starts, and due to the bacteria “climbing up” the aligned BNC fibers, when the bacteria reach the liquid– ATT scans (0.87 ± 0.09 W m−1 K−1 ) resulted twice as large as air interface the typical hydrogel BNC pellicle. The system was cultured for compared to the value obtained using the 3𝜔-method (0.3 ± 0.02 5 days at 30 °C under static conditions until a layer of BNC hydrogel (ran- W m−1 K−1 ). The origin of these different values in the perpendic- dom fiber organization) was detected at the liquid–air interface. A-BNC ular direction possibly arises from different thermal resistances films were obtained within the length of the tube. between parallel fibers. From those results, we foresee the poten- Reuse of the Bacterial Substrates (Agar): The confluence of bacterial tial of natural-based materials, which may outperform polymer colonies of the agar square used as bacterial substrate does not decrease. Therefore, the reuse of this piece of agar, which was carefully released from and synthetic materials in some properties. the produced cellulosic film and confined again at the bottom of a new cul- ture tube filled with fresh liquid culture media (free of bacteria) (Figure 2G) 3. Conclusions was evaluated. The system was cultured for 3 days at 30 °C under static conditions until a layer of BNC hydrogel (random fiber organization) was Inspired by the bacterial cellulose biosynthesis, we presented a detected at the liquid–air interface. A-BNC films were obtained within the facile and reproducible method to obtain films of aligned cellu- length of the tube. Agar substrates were reused for at least 7 cycles, ob- lose fibers (A-BNC) and nonaligned cellulose (BNC) fibers in a taining A-BNC and BNC films in only 3 days. “Film-to-Film” Biosynthesis; Reuse of BNC Film: The uncleaned BNC single step. Bacteria confined at the bottom of a vessel and stati- film produced at the liquid–air interface was detached from A-BNC by cally incubated for a few days biosynthesized BNC nanofibers in pulling off the film with Teflon tweezers and cutting with sterilized scis- a single direction toward the liquid–air interface without entan- sors. Then, it was placed at the bottom of a new culture tube previously glement. Cellulose fibers strongly interact, creating an aligned filled with fresh liquid culture media (free of bacteria). The system was film which alignment is maintained upon different dry-wetting cultured for 3 days at 30 °C under static conditions until a layer of BNC cycles without the use of any additionally additive. The reuse of hydrogel (random fiber organization) was detected at the liquid–air inter- face. This results in films containing 2 circular BNC pellicles bound at the bacterial substrates for at least 7 cycles, allowed to speed and in- extremes of an A-BNC film (a “Film-to-film,” Figure 2J). A-BNC films were crease the production of A-BNC. Optical, chemical, and physical isolated. This process could be used repeated for at least 7 cycles, obtain- techniques evaluated confirmed the alignment of the BNC fibers ing “Film-to-film’s” consistently. superficially and in the whole volume of the film. Mechanical Bacterial Nanocellulose Cleaning: Once produced, A-BNC and BNC properties were not reported, to the fragility and low thickness films were separated by carefully cutting and pulling out from the extremes of A-BNC. However, to facilitate the comparison with reported of the films using Teflon tweezers. After separating the films, the cleaning materials, future work includes the in-depth mechanical evalua- process took place separately. BNC films were soaked and stirred with a magnetic stirring plate in the following steps[46,50] : i) 1:1 Ethanol: Milli- tion of A-BNC films. A-BNC demonstrated to be suitable as a cell Q water solution for 10 min, ii) 40 min in boiling Milli-Q water, and iii) carrier substrate, as fibroblasts adhered and grew. The evaluation two periods of 20 min in 0.1 m NaOH (Sigma-Aldrich) aqueous solution. of the thermal conductivity of A-BNC using two independent ap- Finally, the BNC films were rinsed with Milli-Q water until reaching neu- proaches confirmed a fivefold increase of the thermal conduc- tral pH, autoclaved at 121 °C for 20 min and stored suspended in water tivity in the parallel direction to the alignment axis, 1.63 ± 0.15 in glass vials at room temperature. To avoid entanglement of the aligned W m−1 K−1 , whereas, in the perpendicular direction, the thermal fibers, A-BNC films were gently cleaned with the same steps as BNC but without magnetic stirring. conductivity is 0.3 ± 0.02 W m−1 K−1 . The properties risen by Bacterial Nanocellulose Drying: BNC samples were placed between the alignment encompasses the properties already exploited for two Teflon plates on a horizontal surface and dried at 60 °C for at least celluloses as their easy functionalization, biocompatibility, and 12 h under a 1 kg weight. The thickness of the BNC films of 300 μm in purity, which bring cellulose to be used in an even more signif- wet decreased to ≈20 μm upon drying. As A-BNC films are thinner than icant number of fields, and even join the biomaterials’ group of BNC, and thus more difficult to detach from a surface. Thus, A-BNC films our future and active implants. were dried on top of a Teflon plate on a horizontal surface and without any applied weight at 60 °C for at least 12 h. Neither drying with nor without weight modified the alignment of the A-BNC films (data not shown). Biosynthesis of Aligned BNC (A-BNC) and BNC Films—Atomic Force Mi- 4. Experimental Section croscopy (AFM): A-BNC and BNC films were dried on top of a thin con- Biosynthesis of Aligned BNC (A-BNC) and BNC Films—Biosynthesis of A- ductive Si surface. 10 × 10 μm2 micrographs were obtained using a mod- BNC and BNC films: Komagataeibacter xylinus (K. xylinus) strain colonies ular PM/AFM (Keysight 5500 LS SPM/AFM), using tapping mode. The Adv. Sci. 2022, 9, 2201947 2201947 (10 of 14) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH www.advancedsciencenews.com www.advancedscience.com isotropy distribution of AFM images was computed with MountainView8 an isotropic image without any preferential orientation and 1 represents a (Digital Surf). perfectly aligned distribution image.[31] Contact Angle (CA): 3 mm width and 10±2 μm thick dry A-BNC and Polarization-Resolved Second-Harmonic Generation Microscopy (PSHG): BNC films were fixed flat on a Teflon plate with tape. The surface wettability Semiwet A-BNC and BNC films were stained for 5 min with 5 μL with an and capillarity of the materials were assessed by a contact angle measurer aqueous solution of 0.25 mg mL−1 Brightener 28 (seen as bright blue, exc (KRÜSS Drop Shape Analyzer DSA 100), using the sessile drop method. 365 nm) in Milli-Q water. Then, they were observed under a custom-made 2 μL of Milli-Q water were placed on the BNC surface and, after the water multiphoton microscope system, which works both as a two-photon ex- drop was deposited, CA values were computed over 0.5 for 10 s. citation fluorescence microscope (TPEF) and as a polarization-resolved Dissolved Oxygen (DO) Content: The reagent Leuco-indigo carmine second-harmonic generation microscope (PSHG) (SLN Research Facil- was used to quantify the dissolved oxygen (DO) content in liquid HS cul- ity, Dr. Pablo Loza-Alvarez, Institute of Photonic Sciences, Castelldefels, ture media. The Leuco-indigo carmine method is a simple, rapid, and ac- Spain).[53] The illumination source is a Ti Sapphire oscillator that gener- curate colorimetric procedure for determining small amounts of DO in a ates a linearly polarized laser of a wavelength of 810 nm in a frequency liquid sample (0–50 ppm).[48,49] First, a solution of 3.6 mg mL−1 of Milli- of 76 MHz and a 180 fs pulse. The laser then gets reflected in Galvo mir- Q water containing the powder reagent (Indigo-5,5’-disulfonic acid dis- rors and the rotation of such linearly polarized light is performed by the odium salt, Sigma-Aldrich) and 0.04 mg mL−1 of dextrose (Condalab) was rotation of a 𝜆∕2 plate, to illuminate the sample by different angles of ex- prepared (final volume of 2.5 mL). At the same time, 10 mL of 37.5% of citation, from 0° to 180°. The linear polarization of the incident beam was KOH in water (Sigma-Aldrich) were mixed with 37.5 mL of ethylene glycol evaluated from 0° to 180° in steps of 10° using a lambda/2 plate, taking (Sigma-Aldrich). Both mixtures were added together and refrigerated until frames at each step. After reflection in a dichroic mirror, the beam excites the Indigo-Carmine reagent was reduced entirely. This could be noted due the sample through an inverted microscope objective (28.25X Olympus). to the color change: starting from blue to red, and finally turning yellow Immersion oil was added. By reflection, the TPEF signal is obtained back- (Figure S3A, Supporting Information). Test samples were collected with a ward after reflecting Nikon fluorescence cubes and passing through an syringe from the bottom and the top of the culture media from a 30 cm IR filter. SHG signal is captured by a microscope objective (NA = 1.1) long culture vessel at days 1 and 7 of culture. 0.1 mL of the reduced solu- and acquired after reflecting a dichroic mirror that rejects backscattered tion of Indigo-Carmine was added to a 7.5 mL test sample inside a vacuum laser light and modifies the ellipticity of the polarization states and pass- container. The presence of DO oxidized the reagent and made the solution ing through narrowband and IR filters. Brightfield, TPEF, and PSHG image change color. The variation in color of the dye is directly proportional to stacks were obtained from 0° to 180°, as well as Z stacks. Further analysis the amount of DO present in the sample, which can quantitatively be de- was performed pixel by pixel using the stack options “Z project” and “plot termined by using a color scale (Figure S3B, Supporting Information). The Z-axis profile” from the heat map filtered image stacks in ImageJ. The Ori- DO concentration at different time points (days 1 and day 7) of bacterial entationJ ImageJ plugin[74] was used to assess the orientation coherency culture and different culture locations (top and bottom) was evaluated. of the fibers in the images (Equation (1)). The volume extracted was evaluated with the Leuco-indigo carmine reac- Interferometry: A-BNC and BNC films were dried on top of a glass cov- tion. erslip. 3D images of 450 × 350 × 1.50 μm3 (A-BNC) and 85 × 100 × 1.50 Fourier-Transformed Infrared (FTIR) Spectrophotometry: Dry A-BNC μm3 (BNC) were obtained with a high-resolution dual-core confocal and and BNC films were analyzed with an FT/IR spectrophotometer (Jasco interferometry profilometer (Leica DCM 3D optical profilometer), using a 4700LE). The transmittance spectra collection was performed at 2 cm−1 . “retouching surface” operator. For each spectrum, 32 scans were coadded over the measuring range 400– Scanning Electron Microscopy (SEM): Images were obtained using a 4000 cm−1 . Air was used as blank (machine lid open). The spectra were high-performance SEM (QUANTA FEI 200 FEG-ESEM) without metalliza- processed with the Spectra Manager Suite software to reduce CO2 and tion at EHTs of 5–10 kV and 50 Pa (low vacuum conditions). A-BNC and H2 O noise levels, baseline correction, smoothing and peak find. BNC films were fixed flat on aluminum SEM holders with adhesive carbon Optical Microscopy: The morphology and structure of A-BNC and tape. BNC were assessed using a conventional optical microscope (Olympus Small Angle X-Ray Scattering (SAXS): SAXS is commonly used to eval- RXSITRF 52787, MAB INDUSTRIAL). A retardation slide accessory made uate nanofibers’ anisotropy precisely.[34,38] The scattering measurements of a birefringent material (Olympus U-PO3) was coupled to the optical were performed at the NCD-SWEET beamline at ALBA Synchrotron light microscope to obtain polarized microscopy images. For observing the facility in Cerdanyola del Vallès (Barcelona). A monochromatic X-ray beam change of light passing through the dry A-BNC and BNC films, the po- of 8 keV (𝜆 = 0.154 nm) was set using a Si (1 1 1) channel-cut monochro- larized light direction was kept constant, and the sample was circularly mator. An array of Be lenses was employed to collimate the X-ray beam, rotated in the same plane within a turning center point for 360°, perpen- obtaining a beam size at the sample position of 50 × 150 μm2 (V × H). The dicularly to the incident polarized light beam. Photos were taken every 45°. scattering patterns were recorded using a Pilatus3 S 1M detector (Dectris), Fiber Orientation Analysis: The A-BNC fibers orientation was analyzed which consists of a pixel array of 1043 × 981 (V × H) with a pixel size of 172 using the OrientationJ Java plugin[74] for ImageJ/FIJI (D. Sage, EPFL, 2.0.5 × 172 μm2 . The scattering vector q (defined as q = 4𝜋sin(𝜃)/𝜆, being 𝜃 is version). The OrientationJ structure tensor computes the orientation and the scattering angle and 𝜆 is the X-rays wavelength)[75] was calibrated us- isotropy properties in a local window. The local window is characterized ing Silver Behenate as reference, obtaining a sample to detector distance by a 2D Gaussian function of standard deviation 𝜎, based on the evalu- of 6700 mm. Dry A-BNC and BNC samples were fixed flat on 0.50 cm Ø ation of the structure tensor in a local neighborhood. The parameter 𝜎 metal rings by the edges. Samples were scanned perpendicularly to the (expressed in pixel units) is a critical parameter that determines the scale beam direction, with an acquisition time of 30 s. Azimuthal integration of the analysis. It should have a value roughly close to the structure of of the obtained signal was done using PyFAI,[76] limited by 270° of az- interest (e.g., thickness of the cellulose filaments). The smallest local win- imuthal range due to the intrinsic detector gaps limitations but enough to dow available was 1 px, which was the set value used for this analysis. evaluate the 180° of the twofold fiber symmetry. The azimuthal distribu- From the plugin, the “Color Analysis,” “Distribution,” and “Dominant Di- tion against the maximum value was normalized. The integrated scatter- rection” modes were used to analyze images. OrientationJ was also used ing pattern generated a distribution of the diffraction intensity along the to obtain maximum frequencies from the angle distribution to assess the Debye-Scherrer ring, I(Φ) (arb. u.), versus azimuthal angle, Φ (°). A fitting orientation coherency in several imaging techniques (Equation (1)). of the distributions was performed using the OriginLab (OriginLab Cor- poration) nonlinear function Lorentz equation (Equation (2)) from where ⟨ ⟩ the orientation parameters FWHM, OI, and S (Equations (3)–(5)) could Orientation coherency = 2 cos2 𝛼 − 1 (1) be obtained to quantify the alignment of the BNC nanofibers.[34,38] FWHM stands for the full width of the half-maximum intensity of the where 𝛼 represents the angle between an individual fiber and the average azimuthal profiles from the selected diffraction and it is obtained directly fiber orientation. The coherency ranges from 0 to 1, where 0 represents from the fitting. FWHM ranges from 0° to 180° and low values are cor- Adv. Sci. 2022, 9, 2201947 2201947 (11 of 14) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH www.advancedsciencenews.com www.advancedscience.com related with the higher orientation of the sample.[34,38] OI (also as fc ) is Evaluation of A-BNC and BNC as Cell Carriers: in vitro experiment. A defined as an orientation index and complementary to FWHM. The OI human dermal fibroblast (hDF) cell line (1BR.3.G, ECACC 90020507) was ranges from 0 to 1, with 0 describing a random arrangement and 1 de- used to evaluate the performance of A-BNC and BNC as adherent cell car- scribing a perfect nanofibrils orientation. S is Herman’s order parameter, riers, and how the structuration of A-BNC influences the cell growth. Cells obtained with MatLab using Equations (4)–(6). Similar to OI, a value of S were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) = 0 means the nanofibers are randomly oriented, while S = 1 indicates a supplemented with 2 mm GlutaMax (Gibco), and 10% fetal bovine serum full nanofiber alignment. (FBS; Gibco) at 37 °C in 10% CO2 . The subculture routine for 1BR.3.G consisted of split sub-confluent cultures (70–80%) 1:3–1:6, i.e., seeding at 2A 𝜔 2–4×10 000 cells cm−2 using 0.05% trypsin/EDTA; 10% CO2; 37 °C. hDF y = y0 + ⋅ (2) 𝜋 4(x − xc )2 + 𝜔2 were cultured on dry sterile samples of A-BNC and BNC films. In brief, cel- lulose samples were autoclaved (121 °C, 20 min) and dried on autoclaved 180◦ − fwhm glass microscope slides at room temperature in a laboratory hood for 6 OI = (3) h, and finally irradiated with UV light for 30 min to ensure sterility. Slides 180◦ were placed in Ø 8.5 cm Petri dishes and preconditioned with cell media, 3 cos2 Φc,z − 1 before seeding 1.5 m cells per dish. Cell media (20 mL) was changed every S = (4) 2 days. After 4 and 7 days of culture, images were obtained with an in- 2 verted microscope (Nikon Eclipse Ts2). From optical microscope images, the orientation coherency was measured with the OrientationJ[74] plugin cos2 Φc,z = 1 − 2 cos2 Φ200,z (5) for ImageJ (Equation (1)). ∑𝜋 Statistical Analysis: Quantitative data are expressed as means ± stan- 0 I (Φ) sinffi cos2 Φ dard deviation. Statistical analyzes were performed with Graph Pad Prism cos2 Φ200,z = ∑𝜋 (6) 8 software using one-way ANOVA followed by Tukey’s multiple compari- 0 I (Φ) sinffi son test. Statistical significance was accepted when P-values were ≤0.05 and summarized as * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = Thermal Conductivity: The thermal conductivity, k, of a material is a P ≤ 0.0001 for the calculated P-values. measure of its ability to conduct heat. Thermal conductivity measurements of A-BNC and BNC were per- formed using the 3𝜔-method and a novel contactless frequency-domain Supporting Information approach designed Pérez et al.,[66] and labeled after Anisotropic Ther- moreflectance Thermometry (ATT). The latter methodology is particularly Supporting Information is available from the Wiley Online Library or from suitable to study thermally anisotropic materials (e.g., aligned BNC) since the author. it delivers the angular distribution of the thermal conductivity perpendic- ular to the surface of the sample. Measurements were performed with the described system on A-BNC films dried on silicon wafers at room temper- Acknowledgements ature, to which a gold coating was added. Authors acknowledge the critical reading of Prof. Anna Roig, and the The 3𝜔-method is based on electrically heating a thin planar resistor us- discussions of Dr. Martí Gich and Dr. Mariano Campoy. The also ing an AC harmonic current I0 at a frequency 𝜔, and subsequently measur- acknowledge the funding from: RTI2018-096273-B-I00 and PID2021- ing the resultant voltage drop at the first (V𝜔) and third (V3𝜔) harmonics. 122645O-BI00 funded by MCIN/AEI//10.13039/501100011033/FEDER By defining the normalized temperature coefficient of resistance as Equa- “Una manera de hacer Europa,” PID2019-104228RB-I00 funded by tion (7) with R0 the resistance of the resistor at the temperature T0, the MCIN/AEI/10.13039/501100011033, the Generalitat de Catalunya amplitude of the AC component of the temperature oscillations induced (2017SGR765), the “Severo Ochoa” Programme for Centres of Excellence can be determined as Equation (8). in R&D (SEV-2015-0496 and CEX2019-000917-S (FUNFUTURE)), and Grant No. PID2020-119777GB-I00 (THERM2MAIN). SAXS experiments 𝛽 ≡ (1∕R0) [𝜕R∕𝜕T] (7) were performed at NCD-SWEET beamline at ALBA synchrotron (Cerdany- ola del Vallès, Spain) with the collaboration of ALBA staff. PSHG imaging ΔTAC = 2 |V3 𝜔 |𝛽| V 𝜔 | (8) was performed at the SLN Research Facility, Dr. Pablo Loza-Alvarez, Institute of Photonic Sciences (ICFO) (Castelldefells, Spain). Cell culture By solving the 2D heat equation for the geometry of a linear heat source was performed at the Cell Culture facility of the University Autonomous supported on a semi-infinite medium, the thermal conductivity 𝜅 of such of Barcelona (SCAC-UAB). The authors acknowledge the use of Bioren- medium can be obtained as Equation (9). der.com. Nanthilde Malandain (Ph.D. scholarship PRE2019-089754) is enrolled in the Ph.D. program of Biotechnology of the UAB. 𝜅 = −P02𝜋l1 (𝜕ΔTAC∕𝜕 ln 2𝜔) (9) With P0 the total dissipated power at the resistor and l the length of Conflict of Interest the resistor. Since the AC frequency √ determines the thermal penetration The authors declare no conflict of interest. depth according to 1∕q = 𝛼∕i2𝜔, for thick films supported on a semi- infinite medium (i.e., a substrate) the ΔTAC versus ln 2𝜔 curve shows low and mid-frequency regimes that primarily correspond to the substrate and Author Contributions the supported film, respectively. Obtained from the slope (𝜕ΔTAC∕𝜕 ln 2𝜔) of both regions, this method is straightforward to determine the thermal N.M.: Methodology, Validation, Formal analysis, Investigation, Visual- conductivity. ization, Writing – original draft, review & editing. S.R.-S.: Methodology, Statistics were performed on 7 different resistors thermally evaporated Writing – editing. I.A.-S.: Methodology, Writing – editing. N.M.: Investi- on BNC films yielded to obtain the thermal conductivity. gation, Visualization, Writing: editing. K.X.: Investigation, Visualization, Transmission electron microscopy (TEM). A-BNC and BNC films were Writing: editing. E.S.: Resources, Investigation, Visualization, Writing: re- dried on top of a copper TEM grid. Images were obtained using a high view & editing. J.S.R.: Resources, Investigation, Visualization, Writing: angular range TEM (JEOL 1210 TEM), operating at 120 kV with an ORIUS review & editing. A.L.: Conceptualization, Funding acquisition, Supervi- 831 SC 600 Gatan camera. SAED was used to obtain the diffraction pattern sion, Methodology, Validation, Formal analysis, Investigation, Visualiza- from the A-BNC and BNC films, to assess its crystallinity. tion, Writing – original draft, review & editing. Adv. Sci. 2022, 9, 2201947 2201947 (12 of 14) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH www.advancedsciencenews.com www.advancedscience.com Data Availability Statement [27] H. Gwon, K. Park, S.-C. Chung, R.-H. Kim, J. K. Kang, S. M. Ji, N.-J. Kim, S. Lee, J.-H. Ku, E. C. Do, S. Park, M. Kim, W. Y. Shim, H. S. 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