# In situ multiscale characterization of cellulose nanocrystals orthotropic organization achieved by combining ultrasound and frontal ultrafiltration

F. Bosson, W. Chèvremont, M. Karrouch, D. Blésès, V. Delplace, N. Hengl, F.

Pignon

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## To cite this version:

F. Bosson, W. Chèvremont, M. Karrouch, D. Blésès, V. Delplace, et al., In situ multiscale characterization of cellulose nanocrystals orthotropic organization achieved by combining ultrasound and frontal ultrafiltration. Carbohydrate Polymers, 2025, 362, pp.123680. 10.1016/j.carbpol.2025.123680. hal-05327752

HAL Id: hal-05327752

https://hal.science/hal-05327752v1

Submitted on 23 Oct 2025

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# In situ multiscale characterization of cellulose nanocrystals orthotropic organization achieved by combining ultrasound and frontal ultrafiltration

F. Bosson<sup>a</sup>, W. Chèvremont<sup>b</sup>, M. Karrouch<sup>a</sup>, D. Blésès<sup>a</sup>, V. Delplace<sup>c</sup>, N. Hengl<sup>a</sup>, F. Pignon<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Cellulose nanocrystals
Frontal ultrafiltration
Ultrasound
Orthotropic
SAXS
SALS

## ABSTRACT

Combining frontal ultrafiltration (FU) and ultrasound (US) processes, an orthotropic organization of cellulose nanocrystals (CNCs) suspensions was achieved and characterized at multiscale, from the nanometer length scale by small angle X-ray scattering (SAXS) and dichroism, to the micrometer length scale by small angle light scattering (SALS). A dedicated channel cell has been developed to simultaneously generate a vertical acoustic force via a vibrating blade at the top and a transmembrane pressure force through the membrane at the bottom. Three specific orientations in three successive layers were revealed in a one-step FU-US process. In a first layer at the bottom, the CNCs were concentrated under the action of transmembrane pressure with their director aligned parallel to the horizontal membrane surface. In a second upper layer a random orientation of the CNCs was detected. Finally, in the upper part of the channel, a third layer of CNCs with their director vertically oriented was revealed. Near the membrane surface, transmembrane pressure forces dominate, leading to highly concentrated CNCs deposition with enough consistency to avoid any change in orientation induced by the US at the top of the channel. At increasing distances from the membrane, acoustic radiation forces become predominant, reorienting the CNCs vertically.

## 1. Introduction

Cellulose is the polysaccharide responsible for the hierarchical structure of all green plants, providing them with mechanical strength in the event of external stress or during growth (Martin, 2015; Schütz et al., 2020). Cellulose offers significant advantages over other polysaccharides and inorganic materials, such as material reinforcement, gas permeation Boufi et al., 2014; De Menezes, J., Siqueira, Curvelo, & Dufresne, 2009), optical properties (Poisson & Zhang, 2024) and thermal stability (Lin et al., 2012). Through an acid hydrolysis process using sulfuric acid (H₂SO₄), the amorphous regions of the cellulose are dissolved, leaving only the crystalline parts, known as cellulose nanocrystals (CNCs) (Pandi et al., 2021; Sun et al., 2016). In particular, CNCs are known for their high mechanical properties: the axial elastic modulus of CNCs ranges from 110 to 220 GPa, while their tensile strength is of the order of 7.5 to 7.7 GPa (Domingues et al., 2014). CNCs also have the ability to form helical structures, both in plants and in aqueous dispersions (Martin, 2015; Schutz et al., 2015). Indeed, the presence of surface charges, due to the acid hydrolysis process, enables

CNCs to exhibit lyotropic liquid crystal behavior. It has been demonstrated that there is a critical concentration above which aqueous CNC suspensions spontaneously separate into an isotropic upper phase and a birefringent anisotropic lower phase (Frka-Petesic et al., 2023; Mandin, Metilli, Karrouch, Lancelon-Pin, et al., 2024; Pignon et al., 2024; Voisin et al., 2023; Wang et al., 2016). In the latter, the CNCs are organized in the form of nematic liquid crystals, whose director vector representing the orientation of the CNCs rotates at a constant angle around the cholesteric axis (Martin, 2015). As a result, cellulose is currently the focus of a great deal of research because of its many properties, in addition to its abundance and low cost (Dufresne, 2017).

CNCs, which are the elementary bricks used in this study, are used in the biomedical field, for example as wound healing materials (Hivechi et al., 2020; Shan et al., 2019), biomedical implants (Asadi et al., 2021), or drug delivery systems (Afrin et al., 2022; Akhlaghi et al., 2013; Yusefi et al., 2022). This material is also of interest in the field of tissue engineering, thanks to its cytocompatibility, mechanical properties and bioactivity (Domingues et al., 2014). CNCs have thus been used in tissue engineering of skin (Bacakova et al., 2019), vascular tissue engineering

<footnote><sup>a</sup> Univ. Grenoble Alpes, CNRS, Grenoble INP (Institute of Engineering Univ. Grenoble Alpes), LRP, F-38000 Grenoble, France</footnote>

<footnote><sup>b</sup> ESRF, The European Synchrotron, F-38043 Grenoble, France</footnote>

<footnote><sup>c</sup> Nantes Université, Oniris, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229, F-44000 Nantes, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: frederic.pignon@univ-grenoble-alpes.fr (F. Pignon).</footnote>
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Schematic representation of the flow diagram of the FU-US system. (1) CNC suspension reservoir, (2) syringe, (3) inlet valve, (4) outlet valve, (5) T-junction, (6) pressure sensor, (7) compressed air supply connected to the T-junction, and (8) small container for collecting the permeate, initially filled with deionized water using syringe (9).</figcaption></figure>

(Arias et al., 2018), cardiac tissue engineering (Batta-Mpouma et al., 2021; Ma et al., 2022), liver tissue engineering (Wu et al., 2018), bone tissue engineering (Ali et al., 2022; Hong et al., 2021; Nizan et al., 2020; Osorio et al., 2019), and cartilage tissue engineering (Liu et al., 2022; Luo et al., 2018).

Among these different tissue engineering approaches, one of the current difficult challenge to achieve is to reproduce the complex orthotropic structure of articular cartilage, which consists of three depth-dependent layers. The superficial zone, the intermediate zone and the deep zone, representing respectively 10–20 %, 40–60 % and 30 % of the total cartilage thickness (between 2 and 4 mm) (Brody, 2015; Fox et al., 2009; Lesage et al., 2022). The superficial zone is characterized by collagen fibers aligned parallel to the joint surface near the bone. In the intermediate zone, the collagen fibers adopt an oblique orientation. Finally, in the deep zone, the collagen fibers are oriented perpendicularly to the joint surface.

Some previous works have succeeded to reproduce independently the structural organization of these three orthotropic layers which constitute the articular cartilage. Different strategies have been employed to achieve this goal. For instance, some have used hydrogels with horizontally, vertically, and randomly oriented pores, chemically bonded to mimic cartilage structure, though with insufficient interlayer adhesion under tensile stress (Liu et al., 2021). Other researchers have explored additive manufacturing. A multilayer structure with varying compressive modulus was created using 3D printing of bioinks, but the orientation of the particles within the matrix was not demonstrated (Golzar et al., 2023). In addition, multilayer scaffolds have been fabricated by assembling CNC/PLA layers via liquid-liquid TIPS and solvent-assisted bonding, reproducing cartilage-like properties (Camarero-Espinosa et al., 2016). More recently an original method combining the effects of frontal ultrafiltration (FU) and ultrasound (US) have been developed and have demonstrated the ability to organize CNC suspensions in three successive layered with an orthotropic organization mimicking the one of the articular cartilage (Pignon et al., 2024). However, only one CNC concentration and one acoustic radiation force was tested, based on a single nanometric length scale approach by SAXS.

The aim of the present work is to enlarge the knowledge acquired in this first result (Pignon et al., 2024) with an original multiscale approach combining in situ SAXS, in situ SALS and in situ dichroism measurements during the FU-US process, in order to probe the time dependent structural CNCs orientations and organizations, from the nanometer size

of the CNCs to micrometer scale corresponding to their cholesteric arrangement. In addition, an exhaustive experimental study exploring the effect of different acoustic radiation powers, and different initial CNC concentration, has been conducted.

To reproduce this orthotropic structure, a channel-type filtration cell combining FU and US was developed (Fig. 1), as mentioned previously (Pignon et al., 2024). The filtration cell was designed to generate a US pressure wave that induces a vertical acoustic radiation force, thanks to a vibrating ultrasonic blade placed at the top of the channel, and a transmembrane pressure force through a membrane designed for FU and located at the bottom of the channel. The vertical acoustic radiation force generates an acoustic streaming in the filtration cell. A supplementary original characterization using dichroism is proposed for a better understanding of the time dependent evolutions of the structure under in situ FU-US process. Dichroism measurements eliminate the need to use large instruments to interpret changes in organization at the particle level, which is an undeniable advantage in terms of implementation. Dichroism was used to characterize the particle orientation angle, which could be compared with the results obtained by SAXS.

## 2. Materials and methods

### 2.1. Preparation of CNC suspensions

CNCs were purchased from the UMaine Development Center (University of Maine, Orono, ME) as an aqueous suspension with a concentration of $C_{CNC} = 10.6$ wt%. The dimensions of the CNCs were measured previously from transmission electron microscopy (TEM) images of negatively stained preparations on a JEOL JEM-2100 Plus microscope operating at 200 kV (Mandin, Metilli, Karrouch, Lancelon-Pin, et al., 2024). 225 particles were measured from the TEM images using ImageJ software (National Institute of Health, USA). The CNCs had a length of $127.4 \pm 34.1$ nm and a width of $10.9 \pm 2.6$ nm, with an aspect ratio of $12.3 \pm 4.3$. The CNC suspension was sonicated using a sonication probe (Branson Digital sonifier, Marshall Scientific, Hampton, NH, 250 W output power). A 200 ml volume of suspension was sonicated by applying a 30 % duty cycle and 0.5 W output control for 10 min, then 5 min. Two suspensions at two different concentrations were prepared by dilution in deionized water ($C_{CNC} = 6$ wt% and $C_{CNC} = 10.6$ wt%). These suspensions were then agitated vigorously for 2 h. The two suspensions at 6 wt% and 10.6 wt% have 83 % and 100 % cholesteric phase, respectively (Mandin, Metilli, Karrouch, Blésés, et al., 2024).

### 2.2. Frontal ultrafiltration cell coupled to ultrasound

A channel-type filtration cell combining FU and US was previously developed (Fig. 1), possessing the same characteristics as those used in previous studies (Jin et al., 2014, 2015; Pignon et al., 2024). Briefly, it was designed to monitor in situ changes in dynamic structural organization in SAXS/SALS. The cell was made of transparent polycarbonate for SAXS and PMMA for SALS and dichroism, with a channel 4 mm thick along x, 8 mm deep along z and 100 mm long along y. A 3 mm thick and 100 mm long vibrating blade in titanium was positioned in the upper part of the cell, at a height $z = 6400$ µm from the surface of the ultrafiltration membrane. The vibrating blade was connected to a sonotrode (Sodeva TDS, Méry, France) consisting of a piezoelectric transducer (20 kHz) attached to a metal rod. A polyethersulfone (PES) filtration membrane (Orélis Environnement, France) with a 100 kDa cutoff was employed in the system.

The CNC suspension was pumped from the reservoir containing the CNC suspension (1) using a syringe (2) to fill the entire channel (Fig. 1). An inlet valve and an outlet valve were placed at the inlet (3) and outlet (4) of the channel, respectively. A T-junction (5) and pressure sensor (6) (FP 110 FGP Sensors & Instrument) were placed between the outlet valve and the filter cell outlet. The upper part of the T-junction was connected to compressed air (7), and the pressure valve protected the
<figure><img src="image_6.png" /><figcaption>Fig. 2. Determination of calibration curve from scattering intensity of CNC suspensions at rest using SAXS measurement. (a) CNC scattering patterns obtained in capillaries, sample-to-detector distance D = 1.5 m. (b) Apparent structure factor, S(q), calculated from form factor P(q) and scattering intensity I(q) for different CNC concentrations. c) CNC calibration curve representing CNC concentration (wt%) as a function of interparticle distance d (nm), which can then be used to calculate the local CNC concentration by measuring interparticle distance d from the SAXS patterns in the filtration cell.</figcaption></figure>

suspension air circuit during channel filling. The permeate was collected in a small container (8). Prior to experiment, the lower compartment of the FU-US cell (permeate) was initially filled with deionized water using syringe (9).

## 2.3. Filtration / ultrasound – In situ SAXS

### 2.3.1. In situ SAXS

SAXS measurements were carried out on the TRUSAXS ID02 beam-line at ESRF, Grenoble (European Synchrotron Radiation Facility). Measurements were carried out at room temperature, with a sample-to-detector distance D of 1.5 m and 10 m, and an X-ray energy of E = 12.232 keV (wavelength λ = 0.10 nm), allowing a scattering wave vector magnitude range, Δq, of (8 × 10⁻³ – 5) nm⁻¹, where q = 4π/λ sin(θ/2) with θ the scattering angle. Using pinhole collimation, a beam

with a cross-section full-width at half maximum measuring approximately 25 µm vertically and 56 µm horizontally at the sample position was produced (Narayanan et al., 2022). The incident beam passes through the sample in the FU-US cell through a window of 3 mm × 5.5 mm × 0.3 mm (width x height x thickness) positioned in the middle of the channel, and the 2D scattering patterns are recorded on a high-resolution pixel array detector (Eiger2-4 M, Dectris). The exposure time was 0.3 s. The scattering patterns were normalized to an absolute scale using the standard procedure (Narayanan et al., 2022). A vertical scan is systematically performed from the membrane surface to the vibrating blade. Scattering patterns are recorded along the entire length of this scan, every 20 µm up to z = 400 µm, then every 200 µm up to the vibrating blade. Data were analyzed as previously described (Jin et al., 2014; Pignon et al., 2011, 2024; Rey et al., 2019). Briefly, the 1D scattering intensity curve I(q) was obtained by azimuthally averaging the normalized scattering patterns. Before experimentations with CNC suspensions, the cell was filled with demineralized water and the scattering intensity of the cell was recorded for each z position. The background scattering intensity was systematically subtracted from the scattering intensity of the CNC suspensions in the filtration cell. Azimuthal averages of the scattering patterns were calculated after correcting for deviations between detection modules using SAXS Utilities software (Sztucki, 2021). Anisotropy analysis was then performed on 2D models using MATLAB Small-Angle Scattering Evaluation Tool (SASET) (Muthig et al., 2013). More specifically, the principal component analysis (PCA) method was calculated from 2D SAXS patterns, which provides anisotropy values ranging from 0 for isotropic suspensions to 1 for fully

aligned systems. The anisotropy and direction of maximum diffusion ψ₀ were calculated via PCA, which was performed over a q-range of 0.02 to 2 nm⁻¹ (Mandin, Metilli, Karrouch, Lancelon-Pin, et al., 2024; Pignon et al., 2024). These calculations allowed the quantification of the impact of filtration and ultrasound on the level of anisotropy and alignment of CNCs at different distance z from the membrane surface.

### 2.3.2. Structure factor and scattering intensity calibration curve for CNCs

In order to determine the CNC concentration in situ and over time in the filtration cell, from the membrane surface to the vibrating blade, a CNC calibration curve was established. To achieve this, different suspensions of CNC at different concentrations were prepared by diluting the mother suspension of CNC from the University of Maine at Cₚₚₐ = 10.6 wt% to obtain a concentration range from 0.1 to 10 wt%. These suspensions were placed in 2 mm diameter capillaries and observed by SAXS at a detector distance of 1.5 m and 10 m (Fig. 2a).

Since scattering patterns are anisotropic, this anisotropy can influence the structure factor at low wave vectors. However, in this structure factor, a peak characteristic of the interparticle distance could be observed at a qₚₐₖₐ wave vector (Fig. 2b) linking it to the mean interparticle distance d. Thus, the CNC form factor P(q) was calculated based on measurements on the lowest concentrated suspension (Cₚₚₐ = 0.1 wt %), i.e., where only the form factor P(q) is represented. As the scattering intensity is proportional to the product of the form factor and the structure factor, by dividing all the I(q) obtained for the CNC suspension range by this form factor, it is possible to calculate the apparent structure factor S(q) (Fig. 2b) (Hilfiker et al., 1990; Singh, 2017). As previously discussed, the q-value corresponding to the peak of S(q), denoted as qₚₐₖₐ in the following, is related to the average interparticle distance d via the relation d = 2π/qₚₐₖₐ. A calibration curve of CNC concentration versus interparticle distance was obtained (Fig. 2c). A power-law fit of these data gave access to the relationship between mass concentration C and interparticle distance d:

$$C = 7990.7 * d^{-2} \qquad (1)$$

or equivalently, d = 89.4 * C⁻⁰.⁵.

The apparent structure factor S(q) was calculated for each scattering spectrum recorded at each position z from the membrane surface under the different operating conditions (Pignon et al., 2024; Semeraro et al., 2020). Based on these apparent structure factors S(q), the corresponding mean interparticle distance d was evaluated. Finally, using Eq. 1, the local CNC concentration was calculated.
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Experimental conditions for filtration time (min), ultrasonic power (%) and transmembrane pressure (x 10⁵ Pa) for the two experiments carried out separately, one after each other, on CNC suspensions at C<sub>CNC</sub> = 10.6 wt% (blue) and C<sub>CNC</sub> = 6 wt% (green). These two separate measurements are plotted on the same graph for comparison of the evolutions during time and different trans-membrane pressures and US power applied. Each point on the graph corre-sponds to a scan *z* measured inside the filtration cell by SAXS. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** SALS device setup and principle. (a) Schematic representation of the SALS device principle, showing the He-Ne laser, neutral filter, sample, Fresnel lens, camera lens, and camera, with the laser beam passing through these components (Pignon et al., 2021) and (b) Photograph of the SALS setup with the FU-US cell, including the He-Ne laser, neutral filter, Fresnel lens, and the FU-US cell in position.</figcaption></figure>

### 2.3.3. Experimental protocol

A CNC suspension at a given initial concentration was introduced into the channel of FU-US cell and positioned on ESRF's ID02-TRUSAXS beamline. This cell generates a US-induced vertical acoustic radiation force at the top of the channel and simultaneously a transmembrane pressure force at the bottom of the channel near the membrane surface, as introduced in previous works (Jin et al., 2014, 2015). After intro-duction of the CNC suspension, a transmembrane pressure (ΔP = 1.2 × 10⁵ Pa) and a given acoustic radiation force (%) are applied

simultaneously, at room temperature, constituting the time $t_f = 0$ of filtration. The maximum acoustic radiation force applied (100 %) cor-responded to an acoustic amplitude $a_{US} = 1.6 \ \mu m$, calculated previously by Jin et al. (Jin et al., 2014, 2015). This corresponds to an acoustic pressure $P = 2\pi\rho\nu f_{US} a_{US} \approx 300 \ \text{kPa}$ and an acoustic power $P_a = \frac{P^2}{\rho\nu} \approx 6 \ \text{W.cm}^{-2}$, where $\rho = 1000 \ \text{kg.m}^{-3}$ is the density of water, $\nu = 1480 \ \text{m.s}^{-1}$ is the speed of sound in water and $f_{US} = 20 \ \text{kHz}$ is the fre-quency of the ultrasonic wave, was calculated previously by Pignon et al. (Pignon et al., 2024). Acoustic radiation forces of 0, 20, 40, 60, 80, and 100 % were applied, corresponding to acoustic powers ($P_a$) of 0, 1.2, 2.4, 3.6, 4.8, and 6 W.cm$^{-2}$, respectively.

During filtration, the filtration cell was moved in front of the X-ray beam at increasing distances *z* from the membrane surface to the vibrating blade, and the scattering patterns were recording along this scan. This *z*-scan was performed regularly at different filtration times ($t_f$) to monitor the evolution of structure formation under the application of pressure and ultrasonic forces. The conditions of pressure, acoustic ra-diation force, and concentration as a function of filtration time are shown in Fig. 3.

## 2.4. Filtration / ultrasound – in situ SALS

### 2.4.1. In situ SALS

The Small Angle Light-Scattering (SALS) device used for the exper-iment was developed and built as previously explained (Piau et al., 1999; Pignon et al., 2021) and upgraded (Pignon et al., 2021), and schematically represented in Fig. 4. Briefly, the device consists of a 2 mW laser beam (He-Ne) with a wavelength of 632.8 nm and a Fresnel lens for collecting the forward-scattered light. The detector is an Allied Vision monochrome digital camera (AV MAKO G-419B POE): CMOS sensor (2048 × 2048 pixels, 11.3 × 11.3 mm²). Image processing was carried out using a video system and a specially developed software (Vimba Matlab) that allowed grouping and averaging of scattering inten-sity with SAXS Utilities (Sztucki & Narayanan, 2007). Scattering patterns were recorded by the camera and video system throughout the experiment, with an exposure time of 0.6 s. The modulus of the scat-

tering vector was calculated using the following equation: $q = (\frac{4\pi n}{\lambda}) \sin(\frac{\theta}{2})$, with $\lambda$, the wavelength of light in the sample; $n$, the refractive index ($n = 1.3$ in water); and $\theta$, the scattering angle. Light scattering measurements covered a range of $q$ from $2 \times 10^{-4}$ to $4 \times 10^{-3}$ nm$^{-1}$. The laser beam spot size was about 250 µm.

### 2.4.2. Experimental protocol

The same ultrasonic filtration protocol employed in SAXS was used in SALS, with identical CNC suspensions. The aim was to compare both measurements to characterize the structural organization of the CNC suspensions at different length scale from micrometric to nanometric one. During the filtration, the cell was moved in front of the laser at increasing distances *z* from the membrane surface to the vibrating blade, and the scattering patterns were recorded along this scan with *z* displacement precision every 250 µm to the ultrasonic blade. The *z*-scan was performed regularly at different filtration time $t_f$, identical to those performed in SAXS in order to monitor the structure formation at the micrometer scale under acoustic and transmembrane pressure forces. In addition to the structural characterization, SALS also has enable the observation of the cholesteric phase within the CNC suspensions. This technique was employed to determine the cholesteric pitch $P_i$ and monitor its evolution under varying experimental conditions, such as filtration time and US forces. Tracking these changes provided valuable insights into how acoustic forces and transmembrane pressure influence the microscopic organization of CNCs.
<figure><img src="image_9.png" /><figcaption>Fig. 5. Top view of the schematic diagram of the dichroism set-up.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Fig. 6. (a) Normalized 2D SAXS patterns of a CNC suspension, recorded in situ from the membrane surface to the vibrating blade at 80 % acoustic radiation force ($P_a = 4.8 \text{ W} \cdot \text{cm}^{-2}$) and $\Delta P = 1.2 \times 10^5 \text{ Pa}$, for an initial CNC concentration of 6 wt% and at filtration time of 94 min. (b) Transposition of 2D SAXS patterns in a corresponding CNC orientation at different distances $z$ from the membrane surface, revealing the orthotropic structure.</figcaption></figure>

## 2.5. Filtration / ultrasound – In situ dichroism

### 2.5.1. In situ dichroism

Dichroism ($\Delta n''$) measurements were performed to determine particle orientation within the FU-US cell. Unlike SAXS, which provides information on nanoscale structure by measuring X-ray scattering, dichroism is more accessible, requiring less equipment, and offers information on particle orientation at different length scales (from nano to micro). Previously used to study the orientation dynamics of lyotropic colloidal suspension of sepiolite clay subjected to extensional flow, this method has enabled to provide the first experimental evidence in a 3D system of the “strong flow-weak flow” phenomenon (Pignon et al.,

2003). For the first time, this method is performed on CNCs to evaluate the structural organization changes at particle length scale using lab scale experimental set-up. Dichroism is the difference between the principal values of the imaginary part of the refractive index tensor $n_{ij}''$. The optical equipment used for the experiments is shown in Fig. 5.

It consists of a Ne–He laser that generates monochromatic light of wavelength $\lambda = 632 \text{ nm}$ (beam spot size of about 250 µm and exposure time of 0.1 s). This light is reflected through a straight prism and then passes through the following elements: a linear polarizer, a photoelastic modulator (PEM), a quarter-wave plate, the filter cell, and a photodiode used as a detector. The linear polarizer is oriented at 45° to the PEM. The quarter-wave plate is parallel to the linear polarizer. The measured
<figure><img src="image_11.png" /><figcaption>Fig. 7. (a) Apparent structure factor $S(q)$ corresponding to the 2D SAXS patterns recorded from the membrane surface to the ultrasonic blade, $C_{CNC} = 6$ wt%, $\Delta P = 1.2 \times 10^5$ Pa, acoustic radiation force = 80% ($P_a = 4.8$ W.cm$^{-2}$) and filtration time $t_f = 94$ min with the $q_{peak}$ shown for the distance $z = 6400$ µm. (b) Concentration of CNC as a function of the distance $z$, from the membrane surface to the ultrasonic blade for the two CNC concentrations at different US power s applied, in steady state conditions.</figcaption></figure>

intensity can be written as follows:

$$I = I_{dc} (1 + 2\cos2\alpha\tanh\delta''\sin\omega t + 2\sin2\alpha\tanh\delta''\cos2\omega t) \quad (2)$$

where $I_{dc}$ is the initial direct intensity going through the sample after passing through the PEM and the quarter-wave plate, and $\alpha$ is the average fiber orientation angle measured with relation to an arbitrary axis. Optical anisotropy $\delta''$ is linked to dichroism by the relation: $\delta'' = \frac{2\pi\Delta n''e}{\lambda}$ where $\Delta n''$ is the dichroism, and $e$ is the thickness of the sample. The signal intensity is sent to two lock-in amplifiers locked to the frequency $\frac{1}{\omega} = 52$ kHz of the PEM (Pignon et al., 2003).

### 2.5.2. Experimental protocol

To complete the data from SAXS and SALS measurements, the same ultrasonic filtration protocol was employed, with identical CNC suspensions. During filtration, the filtration cell is fixed and the laser and the photodiode are moved together along the cell at increasing distances $z$ from the membrane surface to the vibrating blade, and the transmitted intensity passing through the sample in the filtration cell is recorded on the photodiode along this scan with $z$ displacement precision every 250 µm from the membrane surface to the ultrasonic blade. At each distance $z$, from the signal recorded on the photodiode, the initial direct intensity, $I_{dc}$ and $I(\omega)/I_{dc}$ and $I(2\omega)/I_{dc}$ are recorded and extracted by the two lock-in amplifiers locked in to the frequency $1/\omega$. From equation Eq. S6 and Eq. S7 on supplementary data, angle and dichroism are calculated from three intensities, i.e., $I_{dc}$, $I(\omega)/I_{dc}$, and $I(2\omega)/I_{dc}$, recorded for 20 s to get a mean value in steady state. The z-scan is performed regularly at different filtration time $t_f$, identical to those performed in SAXS and SALS, allowing to characterize the structure formation under acoustic and transmembrane pressure forces.

## 3. Results and discussion

### 3.1. Effect of CNC concentration on the typical orthotropic organization revealed by SAXS

Normalized 2D in situ SAXS patterns measured within the FU-US cell, from the membrane surface to the vibrating blade, for 80% acoustic radiation force ($P_a = 4.8$ W.cm$^{-2}$), a transmembrane pressure $\Delta P$ of $1.2 \times 10^5$ Pa, initial CNC concentration of $C_{CNC} = 6$ wt% and a filtration time $t_f$ of 94 min, are shown in Fig. 6.

Three regions can be clearly identified in these patterns (Fig. 6a), corresponding to the change in orientation of the 2D SAXS patterns.

There is a horizontal anisotropic structure near the membrane surface (z1), an isotropic structure at the center of the filtration channel (z2), and a vertical anisotropic structure near the vibrating blade (z3), corresponding respectively to the superficial, intermediate, and deep zones of articular cartilage, as representation is given in Fig. 6b. A similar behavior was revealed in a previous study (Pignon et al., 2024) but for a more concentrated suspension. In this paper, for a CNC concentration $C_{CNC} = 10.6$ wt%, in the z1 zone, the 2D SAXS patterns are anisotropic and elongated along the vertical direction. Under the effect of trans-membrane pressure, a regular orientation of the CNCs with their long axis parallel to the membrane surface is observed, as previously described (Jin et al., 2015; Gicquel et al., 2019; Semeraro et al., 2020) and (Rey et al., 2019). In the z2 zone, the 2D SAXS patterns evolved from slightly anisotropic to isotropic and finally slightly anisotropic with a change in orientation (elongated in the horizontal direction). This intermediate region corresponds to a transition zone where ultrasonic and transmembrane pressure forces equilibrate. Finally, in zone z3, the 2D SAXS patterns are elongated in the horizontal direction, indicating an orientation of the CNCs with their director vertically oriented, perpendicular to the membrane surface.

In order to calculate the CNC concentration inside the different layers, the apparent structure factor $S(q)$ corresponding to the 2D SAXS patterns, recorded from the membrane surface to the ultrasonic blade, in the same operating condition, are shown in Fig. 7a. Each apparent structure factor exhibits a peak at a wave vector value $q$, identified as $q_{peak}$. For each $z$ position, determining the wave vector corresponding to the correlation peak ($q_{peak}$) allows for the calculation of the local concentration of CNC using the calibration curve (Eq. 1). The results obtained are shown in Fig. 7b.

The concentration of CNC increases near the membrane surface, over a thickness of 2000 µm. This phenomenon called fouling is associated with frontal ultrafiltration, which concentrated the particles near the membrane surface. Indeed, all the nanoparticles are retained by the membrane, and only the water passes through its porosity. This observation was also previously observed (Pignon et al., 2024; Rey et al., 2019; Gicquel et al., 2019; Semeraro et al., 2020) in the case of crossflow ultrafiltration. Depending on the feed concentration, the value of concentration reached at the membrane surface changes from 44 wt% to 56 wt% for $C_{CNC}$ of 6 wt% and 10.6 wt%, respectively. Subsequently, the thickness of the concentrated layer stabilizes at 2500 µm at $C_{CNC} = 6$ wt% whereas for $C_{CNC} = 10.6$ wt% a larger one is observed at a value of 2800 µm. These results are of the same order of magnitude, whereas we might have expected a greater thickness for the more concentrated
<figure><img src="image_12.png" /><figcaption>**Fig. 8.** Anisotropy parameter PCA and direction of anisotropy obtained through SAXS data for CNC suspensions at different concentrations: (a) and (b) $C_{CNC}$ = 6 wt %, (c) and (d) $C_{CNC}$ = 10.6 wt%. (a) and (c) show the anisotropy parameter, while (b) and (d) display the direction of anisotropy. These measurements were performed for different US power at a transmembrane pressure $\Delta P$ = $1.2 \times 10^5$ Pa.</figcaption></figure>

suspension. However, these observations are quite logical since the SAXS measurements were not made at the same time, as shown in Fig. 3. For experiments with a feed concentration of 10.6 wt%, the first scattering pattern was measured after 30 min of filtration, compared with 95 min for the 6 wt% suspension. At the lower initial 6 wt% concentration, it takes longer filtration time to obtain the same concentrated layer thickness, and a lower nanoparticle concentration level is reached. When ultrasound is applied into the filtration cell via the vibrating blade, the same phenomena are observed regardless of initial concentration. For a specific initial concentration, as US power increases, a decrease in the concentration level profile is observed. However, there is no effect of US on layer thickness at any power level. This is because the acoustic phenomena generated by low-frequency US, such as cavitation and microstreaming, create local agitation throughout the layer thickness. These hydrodynamic forces lead to resuspension of nanoparticles without total destruction of the layer. Jin et al. showed that, depending on the suspension filtered, the acoustic effects of US could be very different on the accumulated layer on the membrane surface (Jin et al., 2015). In the case of cohesive systems, or macromolecules, persistent clogging was observed, while filtration performance increased due to local agitation in the concentrated layer. These results therefore confirm that, for the two concentrations used in this work, the US interacts with the entire thickness of the layer. It is this balance between pressure and acoustic forces that is responsible for the appearance of this orthotropic structure similar to that of articular cartilage.

Moreover, at increasing filtration time (corresponding to successive decreasing US power applied from 80 to 0 %) as depicted in Fig. 3, the level of concentration and thickness of the deposit has increased

regularly (Fig. 7b). This phenomenon was the same for the two studied initial CNC concentrations at 6 wt% and 10.6 wt%. This small increase in concentration and thickness of the deposit at decreasing US power applied, could be associated to a balance change in the ratio of acoustic and transmembrane pressure forces in the transition zone. At lower US power, the balance between acoustic and transmembrane forces was in favor of transmembrane pressure forces which had an effect to increase slightly the thickness and the concentration of the deposit. This phenomenon was observed for the two different initial concentrations in CNCs at 6 wt% and 10.6 wt% even though the initial conditions and then the structuring of the deposit before reducing the US power was completely different.

## 3.2. Calculation and interpretation of the PCA parameter in SAXS

The observation of measured 2D SAXS patterns reveals the presence of anisotropic structures. To quantify the anisotropy, a Principal Component Analysis (PCA) was conducted on the entire set of 2D SAXS patterns. PCA analysis of the anisotropic two-dimensional patterns allows for quantifying the angle and degree of particle orientation at different distances *z* from the membrane surface. The choice of anisotropy calculation focused on the PCA method is because it does not require modeling the scattered intensity *I*(*q*) of the sample with determination of the form factor and structure factor. The results obtained are depicted in Fig. 8.

For the CNC suspension at $C_{CNC}$ = 10.6 wt%, Fig. 8c illustrates the evolution of anisotropy as a function of distance *z* from the membrane surface to the vibrating blade. For curves representing filtration and US
<figure><img src="image_13.png" /><figcaption>**Fig. 9.** (a) Thickness of the membrane surface zone z1 (blue), intermediate zone z2 (red), and deep zone z3 (yellow) as a function of acoustic power ($P_a$) and filtration time obtained for a CNC suspension subjected to a transmembrane pressure ($\Delta P = 1.2 \times 10^5$ Pa) at a concentration $C_{CNC} = 6$ wt%, with the 2D SAXS patterns obtained for $P_a = 0$ W.cm$^{-2}$ (left) and $4.8$ W.cm$^{-2}$ (right) at the same corresponding conditions. (b) Variation of the CNC concentration in zone z1 as a function of the distance z from the membrane surface, for an initial CNC concentration $C_{CNC} = 6$ wt% subjected to different acoustic pressures and transmembrane pressure ($\Delta P = 1.2 \times 10^5$ Pa), for different filtration times. (c) Zoom on the end of the deposit between 1000 and 3000 µm, corresponding to the stabilization of the concentration: 2000 µm for $P_a = 4.8$ W.cm$^{-2}$ and $t_f = 94$ min, and 2400 µm for $P_a = 1.2$ W.cm$^{-2}$ and $t_f = 109$ min. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
conditions, the anisotropy calculation on this suspension has revealed three distinct zones: a first zone z1 near the membrane surface where anisotropy was high (up to approximately 1800 µm), a second zone z2 between 1800 and 2400 µm where the anisotropy parameter has dropped to almost zero, and finally a third zone z3 starting from 2400 µm where the anisotropy parameter has increased again. There was also a decrease in the anisotropy parameter near the membrane surface, possibly due to the presence of local agitation near the membrane caused by ultrasound (Pignon et al., 2024). The anisotropy has increased up to 400 µm, then decreased and has fluctuated over the remaining height of the cell towards the membrane surface. This evolution of anisotropy could be explained by the effect of acoustic streaming generated by US (Wiklund et al., 2012). Studied for over half a century, the acoustic streaming is a macroscopic flow caused by a sound field, which exists in a viscous fluid (Leighton, 1995, 2007; Nyborg, 1982). The state of the volume elements of a fluid, in which an acoustic wave propagates, is described by pressure and velocity oscillations. In an ideal fluid, the time-averaged displacement of particles, i.e. the net fluid flow, is equal to zero everywhere. However, in real fluids, viscous attenuation causes the net displacement of fluid particles during each oscillation cycle to become different to zero. This local effect can lead to the global formation of continuous flows (Wiklund et al., 2012). This phenomenon leads to the formation of rotating streaming vortices in the suspension, which can influence the orientation of CNCs. Near the ultrasonic blade, a small vortex has formed, followed by a larger one that has oriented the CNCs perpendicularly to the filtration membrane. Typically, in a closed environment, another large vortex and a small vortex would appear below the first two. However, in our case, the presence of the filtration membrane, which has acted as a porous and non-closed medium, has prevented the formation of these additional vortices. Nevertheless, there was still some local agitation near the membrane surface, which has enhanced filtration and has increased the CNC concentration near the membrane.

For the CNC suspension with $C_{CNC} = 6$ wt%, Fig. 8a illustrates the three distinct zones that are also present, but this time without the decrease in PCA near the membrane, unlike the suspension with $C_{CNC} = 10.6$ wt%. This second suspension is less concentrated and forms less cholesteric phase (83 % compared to 100 % for the suspension with $C_{CNC} = 10.6$ wt%), which could explain this difference. Moreover, the anisotropy level is lower than that obtained with the more concentrated suspension. Indeed, at low concentration, the nanoparticles are more mobile, leading to reduced structural organization. As the concentration increases, the CNCs come closer together, inducing stronger interactions and resulting in better structuring, thus increasing the anisotropy.

Moreover, the orientation angle of the anisotropy varies depending on the position z relative to the membrane surface. Fig. 8b and Fig. 8d present the variation of the orientation angle of the scattering pattern as a function of distance z to the membrane surface for different acoustic radiation forces. It is observed that ultrasound induced a change in CNC orientation from 90° to 180°, reflecting the change in nanoparticle structuring in the filtration channel. Near the membrane surface (angle = 90°), the CNCs are positioned parallel to the membrane surface under the effect of transmembrane pressure forces due to frontal ultrafiltration. As one moves away from the membrane and closer to the vibrating blade, the ultrasonic radiation forces induce a change in orientation of the CNCs in the direction of the induced force. Consequently, over a significant thickness, they become oriented perpendicular to the surface of the filtration membrane (angle = 180°). These behaviors are observed for both concentrations and for each acoustic power Pa. The transition between these two orientation states defines a zone in which the structure becomes isotropic. It is interesting to notice that the resulting structure, similar to that of articular cartilage, can be precisely controlled thanks to this process combining ultrasound and trans-membrane pressure.

Based on previous studies (Pignon et al., 2021, 2024), the most probable explanation regarding the origin of CNC orientation (angle =

180°) in the upper part of the cell, is that the acoustic streaming mechanism induces a flow of the suspension in this zone, and thus the CNC orientation. Observations of anisotropy fluctuations near the surface of the vibrating blade supported the argument in favor of an acoustic streaming mechanism (Pignon et al., 2024). This mechanism, triggered by US vibration, provokes the formation of vortices in the viscous boundary layer near the vibrating surface, known as Schlichting and Rayleigh streaming (Wiklund et al., 2012). Theory suggests that the occurrence of the Rayleigh phenomenon requires specific boundary conditions and confined flow, which are fulfilled in our system. In particular, Schlichting or Rayleigh streaming is favored when: $λ>>h>>δ_v$, where $λ$ is the acoustic wavelength, $h$ is the characteristic length scale of the fluid chamber, and $δ_v$ is the viscous penetration depth, which in an oscillating flow is given by $δ_v = \sqrt{2μ/ω}$, where $ω$ is the fluid's kinematic viscosity and $ω$ is the angular frequency of the acoustic wave (Wiklund et al., 2012). In our system, these conditions are indeed fulfilled, as $λ = 7.5 cm>>h = 6400 μm>>δ_v = 53$ to $560 μm$. All this suggests that Rayleigh acoustic streaming is the physical origin of CNC orientation induced by US waves.

### 3.3. Determination of the thicknesses of the different layers

Using the values of the PCA anisotropy parameter for different experimental conditions, it is possible to determine the thickness of each oriented layer (Fig. S2 in supplementary data) of the orthotropic organization. In our study, we set a PCA value of 0.1 as the threshold below which the structure was considered to be isotropic. This threshold is arbitrary but provides a practical reference for our analysis. We compared the thickness of each of the three zones z1, z2, and z3, as a function of filtration time and acoustic radiation force, for the two concentrations of CNC. The results obtained for these two CNC concentrations ($C_{CNC} = 6$ wt% and $C_{CNC} = 10.6$ wt%) are shown in Fig. 9 and Fig. S3 in supplementary data, respectively.

#### 3.3.1. Superficial anisotropic zone z1

The superficial zone z1 is depicted in blue in Fig. 9a. It appears that the lower horizontal layer is mostly affected by the time of filtration. The pressure forces leading to a concentrated deposition of CNCs on the membrane surface prevent US from disrupting the layers. Indeed, this layer in which CNCs are oriented parallel to the membrane is formed by frontal ultrafiltration. The variation in thickness is related to different filtration times for each acoustic radiation force: the longer the filtration time, the thicker this layer becomes. This z1 zone also corresponds to the most concentrated one within the filtration cell (Fig. 9b). It can be observed that the concentration of CNC stabilizes at the end of this z1 zone.

#### 3.3.2. Intermediate isotropic zone z2

In the intermediate zone z2, represented in red in Fig. 9a, the PCA anisotropy parameter remains low and below 0.1, while the anisotropy direction $ψ_0$ varies from an angle of 90° to 180° (Fig. 8). This isotropic intermediate zone corresponds to a transition zone where US forces and transmembrane pressure balance out, resulting in isotropic organization. The thickness of this layer remains relatively constant (between 400 and 600 µm for $C_{CNC} = 6$ wt% and between 400 and 800 µm for $C_{CNC} = 10.6$ wt%).

#### 3.3.3. Deep anisotropic zone z3

In the deep zone z3, represented in yellow in Fig. 9a, the acoustic pressure forces are predominant. The 2D SAXS patterns are anisotropic, indicating an orientation of the CNCs perpendicular to the membrane surface. This assertion is confirmed by anisotropy direction $ψ_0$ which is approximately 180° throughout the thickness of this zone. As the acoustic pressure increases from 1.2 W.cm$^{-2}$ to 4.8 W.cm$^{-2}$, the thickness of zone z3 increases from 3200 µm to 4000 µm and from 3200 µm to
<figure><img src="image_14.png" /><figcaption>**Fig. 10.** Comparison of (a) transmitted intensity and (b) particle orientation angle obtained by dichroism as a function of height in the cell from the membrane surface for different US powers for a CNC suspension at $C_{CNC} = 6$ wt%. (c) $I(\omega)/I_{dc}$ and $I(2\omega)/I_{dc}$ signals obtained for the CNC suspension at $C_{CNC} = 6$ wt% subjected to acoustic pressure $(4.8 \text{ W.cm}^{-2})$ and transmembrane pressure $(\Delta P = 1.2 \times 10^{5} \text{ Pa})$, during 1 h of filtration.</figcaption></figure>

3600 µm for an initial CNC concentration of $C_{CNC} = 6$ wt% and $C_{CNC} = 10.6$ wt%, respectively. The variation of the thickness is more pronounced in the case of the CNC suspension of 6 wt% than for the one of 10.6 wt% for increasing acoustic powers, which corroborates the fact that acoustic streaming is the physical phenomenon responsible for the change in structure. In fact, the less concentrated suspension has a lower viscosity (Gicquel et al., 2019), limiting the attenuation of streaming. The result for the more concentrated suspension is a slight variation in thickness as a function of applied acoustic power.

### 3.4. Observation of orthotropic structure via in situ dichroism

#### 3.4.1. Transmitted intensity, orientation angle, and dichroism for the 6 wt% CNC suspension

The same FU-US experiments conducted in SAXS were performed

using in situ dichroism. It is the first time that this methodology was performed to understand the structural organization of CNC in situ and in real time during the filtration. The results of transmitted intensity $I_{dc}$, particle orientation angle, and dichroism obtained at the center of the cell for different distances from the membrane surface and for different US powers were depicted in Fig. 10 for the CNC suspension $C_{CNC} = 6$ wt%. In the presence of US, regarding the transmitted intensity (Fig. 10a), two levels of transmitted intensity increases were observed which has defined two regions where a maximum of transmitted intensity is detected: firstly near the membrane surface, and secondly in the region where the acoustic streaming acts. The first maximum of transmitted intensity (around 1.5 mm) likely corresponds to the end of the deposit formed by ultrafiltration (presented in Fig. 9c). Indeed, the high concentration in the deposit leads to an absorption of the Laser beam due to the concentration level which induce a decrease of the transmitted
<figure><img src="image_15.png" /><figcaption>Fig. 11. (a) Comparison of the calculated concentration gradient obtained in SAXS with the transmitted intensity measured in dichroism for the CNC suspension such as C<sub>CNC</sub> = 6 wt%. (b) Comparison of the PCA anisotropy angle obtained in SAXS and the particle angle obtained in dichroism for the CNC suspension at C<sub>CNC</sub> = 6 wt% for acoustic radiation force of 80 % (Pa = 4.8 W.cm<sup>-2</sup>) and 20 % (Pa = 1.2 W.cm<sup>-2</sup>).</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>Fig. 12. Normalized 2D SALS patterns of a CNC suspension, recorded in situ from the membrane surface to the vibrating blade for different acoustic radiation forces (Pa = 0 to 4.8 W.cm<sup>-2</sup>) and filtration time and ΔP = 1.2 × 10<sup>5</sup> Pa, for an initial CNC concentration C<sub>CNC</sub> = 6 wt%. In green the 2D SALS patterns showing oriented cholesterics before filtration, and in red the 2D SALS patterns showing cholesterics near the membrane surface during filtration. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

intensity towards the membrane surface. The second intensity increase (with a maximum around 4.5 mm) could be associated to the disruption of the cholesteric micrometric structure. At rest or near the membrane surface, this cholesteric structure scattered at the micrometer length scale and reduce the transmitted intensity. At increasing US power, this cholesteric structure was disrupted which has induced an increase of the transmitted intensity clearly evidenced in Fig. 10a. However, when the ultrasound power was stopped (0 %), this second intensity peak was no longer present, probably because the cholesterics have reformed after the end of acoustic streaming and the transmitted intensity has followed the one near the membrane surface where a cholesteric organization was observed. These phenomena will be discussed in more details in the next part corresponding to the SALS measurements.

Then, when considering the particle orientation angle (Fig. 10b), before filtration, in the absence of US power, the CNCs were all oriented in the same direction: parallel to the membrane surface. In fact, when the suspension was injected into the FU-US cell, the CNCs have oriented themselves in the direction of flow, i.e. parallel to the membrane surface. However, in the presence of US power, under ultrafiltration, a change in particle orientation between the lower part of the cell, near the membrane, and the upper part of the cell, near the vibrating blade, of approximately 120° from 3.5 to 4 mm from the membrane surface was observed. Thus, the orthotropic structure, previously observed in SAXS, is also revealed by in situ dichroism measurements. However, unlike SAXS, when US is stopped (0 %), the orthotropic structure was still visible. It is possible that after US cessation, the CNCs organize into oriented cholesteric structures, which was also measured by dichroism. This hypothesis could also be confirmed by observation in SALS.

During the application of acoustic pressure, simultaneous recordings were made of the intensities I(ω)/I<sub>dc</sub> and I(2ω)/I<sub>dc</sub> and the intensity scattered by the sample on the screen. As explained in Pignon et al., 2003, the signals I(ω)/I<sub>dc</sub> and I(2ω)/I<sub>dc</sub> are proportional, respectively, to sin(ωt) and cos(2ωt), and are used to determine the dichroism measurement Δn" using the eq. 3:

$$ \Delta n'' = \frac{\lambda \cdot \pi \cdot e}{2} \text{artanh} \left( \sqrt{\left[ \frac{I(\omega)}{I_{dc}} \right]^2 + \left[ \frac{I(2\omega)}{I_{dc}} \right]^2} \right) \quad (3) $$

with e the thickness of the sample (mm). The signal I(ω)/I<sub>dc</sub> has decreased with distance from the membrane surface. This was related to
<figure><img src="image_17.png" /><figcaption>Fig. 13. (a) Comparison of particle orientation angle obtained in dichroism as a function of height in the cell from the membrane surface during relaxation after US cessation for a CNC suspension at $C_{CNC}$ = 6 wt%, under transmembrane pressure $\Delta P$ = $1.2 \times 10^5$ Pa. (b) Evolution of the $I(\omega)/I_{dc}$ signal as a function of relaxation time, measured at different z positions from the membrane surface ($z$ = 5; 6 and 7 mm) after a step for which the CNC suspension at $C_{CNC}$ = 6 wt% was subjected to acoustic pressure ($4.8 \text{ W.cm}^{-2}$) and transmembrane pressure ($\Delta P$ = $1.2 \times 10^5$ Pa), during 1 h of filtration.</figcaption></figure>

the presence of numerous oriented cholesterics near the membrane due to the formation of deposit under ultrafiltration and the increase in concentration in this zone. The cholesterics absorb the radiation, leading to an increase in dichroism (Fig. 10c). The intensity $I(\omega)/I_{dc}$ has decreased until it has reached the intermediate zone, i.e., the zone where US forces and transmembrane pressure forces coexist. The intensity was then almost constant over the rest of the cell height: the ultrasound has destructured the cholesterics. The same phenomena were obtained with a higher concentration of CNC (10.6 wt%), as can be seen in Fig. S4 in supplementary data when comparing the transmitted intensity and the particle orientation angle for the two different concentrations at different acoustic power ($3.6 \text{ W.cm}^{-2}$ and $4.8 \text{ W.cm}^{-2}$).

### 3.4.2. Comparison of the results obtained in dichroism and SAXS

By comparing the results obtained in SAXS and dichroism, one can observe in Fig. 11a that, the first transmitted intensity $I_{dc}$ peak near the membrane surface obtained in dichroism measurements, indeed corresponds to the end of the deposit created by ultrafiltration. This is in accordance with increase of transmitted intensity when the suspension is less concentrated.

Moreover, Fig. 11b compares the particle orientation angles obtained in SAXS through PCA analysis and in dichroism. A change in particle orientation angle is observed regardless of the method used to visualize it. However, it is noted that the change in orientation does not completely coincide between SAXS and dichroism. This is likely due to the higher precision of SAXS, which uses a smaller beam size (25 µm vertically and 56 µm horizontally for X-rays) compared to dichroism, where the laser beam diameter is approximately 250 µm. Thus, the origin ($z$ = 0 mm), defined as the surface of the membrane, is not defined with the same precision between SAXS and dichroism, which may cause a discrepancy. Nevertheless, this second observation by dichroism measurements is very useful as it allowed to follow the same changes observed by SAXS in terms of orientation of the CNCs at a lab scale, in-situ and during different conditions of filtration or acoustic power.

### 3.5. Observation of orthotropic structure via in situ SALS

In situ SALS experiments were conducted under the same conditions as the FU-US experiments used for SAXS and dichroism. This method

was previously used to observe the cholesteric phase of CNC during ultrafiltration (Mandin, Metilli, Karrouch, Bléses, et al., 2024) and under shear and relaxation (Pignon et al., 2021). 2D SALS patterns were obtained at different filtration times under various acoustic radiation forces for the CNC suspension, $C_{CNC}$ = 6 wt%, as shown in Fig. 12.

The green region represents the oriented cholesteric phase before filtration. When placed in the filtration cell, the CNCs align along the direction of shear, forming oriented cholesteric structures perpendicular to the shear direction. In contrast, the red region represents the cholesteric phase in the deposit near the membrane surface under filtration and ultrasound, as shown in previous SAXS and SALS measurement (Fig. S5) (Pignon et al., 2021). Cholesterics structures were observed up to nearly 1500 µm from the membrane surface, consistent with the end of the deposit calculated using SAXS measurements.

However, closer to the membrane, isotropic patterns were observed, indicating the presence of a cholesteric phase (with a pitch) under the combined effects of filtration and ultrasound. This behavior differs from the isotropic patterns observed without ultrasound, raising questions about the impact of acoustic forces near the membrane surface. Between 1500 and 2500 µm, anisotropic patterns without a pitch are observed, indicating the presence of oriented tactoids with CNCs parallel to the membrane. As the distance from the membrane increases, the tactoids have fragmented due to the strong velocity gradient imposed by the ultrasound in the suspension. This fragmentation has led to the disappearance of orientation and the lack of observable structures, particularly near to the membrane surface. Nevertheless, this cholesteric structure was no longer visible in the upper part of the filtration cell, due to the presence of the ultrasound.

At higher CNC concentrations ($C_{CNC}$ = 10.6 wt%), this transition involving tactoids fragmentation was not observed (Fig. S5). This can be attributed to the absence of a Newtonian plateau at a shear gradient of approximately $1 \text{ s}^{-1}$, as shown in the flow curve from (Gicquel et al., 2019). For $C_{CNC}$ = 6 wt%, however, no significant differences are observed between the various ultrasound powers. This is likely because the system has rapidly changed from the cholesteric to the nematic phase, especially given the limited spatial resolution of the measurements ($\pm$ 250 µm). These observations aligned with the hypothesis that the velocity gradient plays a critical role in structuring CNC suspensions under ultrasound.
<figure><img src="image_18.png" /><figcaption>**Fig. 14.** Normalized 2D SALS patterns of a CNC suspension ($C_{CNC}$ = 10.6 wt%), recorded in situ from the membrane surface to the vibrating blade, under filtration ($\Delta P$ = $1.2 \times 10^5$ Pa), immediately after the ultrasound was stopped (left) and 10 min after the ultrasound was stopped (right), after the CNC suspension was subjected to transmembrane pressure ($\Delta P$ = $1.2 \times 10^5$ Pa) and different acoustic pressure, according to the protocol presenting in Fig. 3.</figcaption></figure>

### 3.6. Observation of orthotropic structure relaxation via in situ dichroism and SALS

After the US stopped, the change in particle angle orientation remains visible in dichroism in the upper part, which was not the case in SAXS. In order to understand if the orthotropic structure is maintained

few minutes after US cessation or if the dichroism signal evolution is related to the restructuration of cholesterics, SALS and dichroism measurements were carried out when US was switched off. Relaxation was observed in dichroism for the 6 wt% CNC suspension. Every ten minutes after US cessation, a vertical scan was performed. The results obtained (particle orientation angle) are shown in Fig. 13. Ten minutes after US cessation, the change in particle orientation was still observed. However, starting from twenty minutes after US cessation, the change in particle orientation as a function of z distance from values around 180° to 90° was not observable anymore, indicating that the orthotropic structure was no longer visible. The orientation angle seems to homogenize in the whole cell, which could be attributed to the cholesteric reformation associated to regular rotation of the CNCs orientation (Fig. 13a). This relaxation phenomenon would be studied in more detail to have a clear interpretation of the particle orientation angle in this case of cholesteric reformation which induce complex reorganizations and orientation changes of CNCs at different length scales from nanometer to micrometer ones.

To better understand the relaxation phenomena, $I(\omega)/I_{dc}$ signals were measured over time, after ultrasound had stopped, at different distances z from the membrane surface (Fig. 13b). The relaxation as a function of time is presented after a step for which the CNC suspension at $C_{CNC}$ = 6 wt% was subjected to acoustic pressure of $4.8 \text{ W.cm}^{-2}$ and transmembrane pressure ($\Delta P$ = $1.2 \times 10^5$ Pa) during 1 h. The $I(\omega)/I_{dc}$ signal has decreased as a function of relaxation time after the US stopped and stabilized after 20 min. Cholesterics seem to have reformed rapidly after US was stopped, which can be confirmed by SALS experiments.

The same dichroism experiment was also conducted using SALS to observe the reformation of the cholesteric phase near the US blade after US cessation. Particularly at the higher CNC concentration ($C_{CNC}$ = 10.6 wt%), horizontal formation of cholesterics was observed in the upper part of the filtration cell after the ultrasound was stopped ($t_f$ = 62 min), as shown in Fig. 14. This suggests that while the ultrasound was active, the CNCs were oriented vertically as shown by SAXS measurements, and upon cessation of ultrasound, the vertically aligned CNCs reorganized to form cholesterics with theirs helical axes oriented namely horizontally in the middle of the cell at z = 5000 µm (the SALS patterns exhibit a peak position of the cholesteric structure oriented namely horizontally). Just 10 min after the ultrasound was stopped ($t_f$ = 72 min), cholesterics were present along the entire height of the filtration cell, from the membrane surface to the vibrating blade. This indicates that the cessation of ultrasound allows the CNCs, initially aligned by acoustic forces, to reorganize into their preferred cholesteric structures, suggesting a strong influence of the ultrasound on the orientation dynamics and structural phase formation (from isotropic to cholesteric) of the CNC suspension. A crucial next step would be to stabilize this structure to prevent its reorganization after the US cessation. One potential approach could be the incorporation of a photocurable polymer, allowing the orthotropic structure to be fixed through UV-curing.

### 4. Conclusions

In conclusion, the application of US waves combined with frontal ultrafiltration has enabled the structuring and orientation of CNC suspensions in orthotropic organization at two different concentrations (6 wt% and 10.6 wt%). This structure has been observed in situ for the first time at various scales, from the nanometer length scale (SAXS and dichroism) to the micrometer length scale (SALS). In situ SAXS measurements have highlighted the typical orthotropic structure of cartilage for a given filtration time. Calculation of the PCA anisotropy parameter has provided access to the thickness and orientation level of each obtained layer, which exhibit different orientation angle properties. The effect of US power on the thickness of the typical three layers of the orthotropic structures, have been emphasized by in situ SAXS and related to the equilibrium between the acoustic and transmembrane forces generated by the original set-up and FU-US filtration cell developed. These results
suggest also that acoustic streaming is the physical phenomenon
responsible for the orientation of CNCs in the zone where acoustic forces
are predominant. For the first time, in situ dichroism measurements on
CNC suspensions have complemented the in-situ SAXS experiments,
notably observing the maintenance of the orthotropic structure for a few
minutes after US cessation. Original in situ time resolved SALS mea-
surements have finally confirmed hypotheses regarding the presence of
cholesterics near the membrane surface and their restructuring time
evolutions near the US blade after US cessation. These findings open new
perspectives for the design of orthotropic cellulose biomimetic mate-
rials, particularly for applications in cartilage tissue engineering.

CRediT authorship contribution statement

**F. Bosson:** Writing – review & editing, Writing – original draft, Visualization, Investigation, Formal analysis, Data curation. **W. Chèvremont:** Validation, Investigation. **M. Karrouch:** Validation, Conceptualization. **D. Blésés:** Validation, Conceptualization. **V. Del-place:** Writing – review & editing, Validation, Formal analysis. **N. Hengl:** Writing – review & editing, Validation, Investigation, Formal analysis. **F. Pignon:** Writing – review & editing, Validation, Project administration, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

Acknowledgment

We would like to thank Theyencheri Narayanan (ESRF, Grenoble) for
fruitful discussions and for his kind help in scattering experiments. We
thank Frédéric Hugenell and Eric Faivre (Laboratoire Rhéologie et
Procédés) for their technical assistance. ESRF is acknowledged for pro-
vision of synchrotron beamtime (proposal SC-5319). The data are
available at https://doi.esrf.fr/10.15151/ESRF-ES-1030392887. The
author(s) acknowledge(s) the support of the French Agence Nationale de
la Recherche (ANR), under grant ANR-20-CE43-0015 (ANISOFILM).
LRP is part of LabEx Tec21 (Investissements d'Avenir - grant agreement
#ANR-11-LABX-0030) of Institut Carnot PolyNat (Investissements
d'Avenir - grant agreement #ANR-16-CARN-0025-01), and the Gly-
co@Alps programme (Investissements d'Avenir - grant agreement
#ANR-15-IDEX-02).

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.carbpol.2025.123680.

Data availability

Data will be made available on request.

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