# Role of liquid crystal behavior of CNC/PEGDA/collagen suspensions on their orthotropic organization obtained by ultrafiltration and ultrasound

F. Bosson, A. Laviron, M. Karrouch, L. Metilli, N. Hengl, V. Delplace, F.

Pignon

► To cite this version:

F. Bosson, A. Laviron, M. Karrouch, L. Metilli, N. Hengl, et al., Role of liquid crystal behavior of CNC/PEGDA/collagen suspensions on their orthotropic organization obtained by ultrafiltration and ultrasound. Carbohydrate Polymer Technologies and Applications, 2025, 12, pp.101015. 10.1016/j.carpta.2025.101015. hal-05327775

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# Role of liquid crystal behavior of CNC/PEGDA/collagen suspensions on their orthotropic organization obtained by ultrafiltration and ultrasound

F. Bosson<sup>a, ∗</sup>, A. Laviron<sup>a</sup>, M. Karrouch<sup>a</sup>, L. Metilli<sup>a, †</sup>, N. Hengl<sup>a</sup>, V. Delplace<sup>b, †</sup>, F. Pignon<sup>a, ‡</sup>

ARTICLE INFO

Keywords:

Cellulose nanocrystals
PEGDA
Collagen
Dichroism
Rheometry
Colloid
Liquid crystal

A B S T R A C T

This study explored the effect of adding biologically relevant macromolecules on the preservation of cellulose nanocrystal (CNC) orthotropic organization already obtained in aqueous media, via the combined action of frontal ultrafiltration (FU) and ultrasound (US). In particular, the effect of a photocurable polymer poly(ethylene glycol) diacrylate (PEGDA) with aqueous CNC suspension, in 80/20 weight ratio [water/PEGDA] and collagen were explored. Rheometric and polarized light microscopy measurements were used to reveal the liquid crystal behavior of suspensions. In situ dichroism during the FU/US process allowed to examine the conservation of orthotropic organization. First, the results have proved that the liquid crystal-like behavior is essential for achieving the desired orthotropic orientation of CNC/PEGDA/Collagen suspensions under FU and US. Second, acoustic power and filtration time have been identified as key parameters in preserving the orthotropic arrangement. Third, specific formulation conditions have been found to successfully achieved the orthotropic structure: i) without collagen, 20 wt% PEGDA and 5 to 8 wt% CNC; and ii) with collagen at acidic pH, 20 wt% PEGDA, 6 to 8 wt% CNC, and 0.01 to 0.1 mg/mL collagen. These findings pave the way for future investigations to produce orthotropic hydrogels for tissue engineering, by combining FU/US process with photocrosslinking.

1. Introduction

Cellulose, the most abundant polysaccharide in nature, provides mechanical support in plants and has gained significant interest in biomaterials research (Martin, 2015; Schütz et al., 2020). Nano-celluloses are particularly interesting building blocks for the design of innovative biosourced materials with numerous potential applications. An exponentially increasing number of studies are devoted to understanding and controlling the structural organization of these nano-celluloses (Hickey & Pelling, 2019; Schütz et al., 2020; Thomas et al., 2018; Voisin et al., 2023) and the way they are chemically derivatized and processed, in order to achieve specific functional properties in various industrial applications (Tang et al., 2017). Derived from cellulose through acid hydrolysis, cellulose nanocrystals (CNCs) exhibit remarkable mechanical properties, with an axial elastic modulus ranging from 110 to 220 GPa and a tensile strength between 7.5 and 7.7 GPa, making them ideal as reinforcing agents for scaffold materials (Domingues et al., 2014; Pandi et al., 2021; Sun et al., 2016). Beyond their high strength, CNCs exhibit liquid crystal behavior in aqueous solutions, CNCs can self-organize into anisotropic phases such as nematic and cholesteric liquid crystal. This property enhances their potential for alignment and orientation in biomaterials (Frka-Petesic et al., 2023). One of the interesting features of this behavior is that CNCs

maintain a constant distance between them regardless of the external conditions to which they are subjected. If these rods are subjected to a flow, they will naturally all orient themselves in the direction of the flow velocity, forming a nematic organization. In the same way, if they are pushed towards the surface of a filtration membrane by a pressure force, they will tend to orient themselves parallel to the surface of the membrane (Mandin et al., 2024; Pignon et al., 2024). From a macroscopic point of view, this liquid crystal-like behavior is revealed in terms of the evolution of the viscosity as a function of the shear rate with the typical three regime behavior (Lagerwall et al., 2014; Mitov, 2017; Pignon et al., 2021).

However, when CNCs are used as nanofillers mixed with polymers in nanocomposite materials, the control of their organization and orientation at a macroscopic length scale remains a key challenge that was only tackled by a few research groups (Dumanli & Savin, 2016; Mandin et al., 2024; Metilli et al., 2025). In this general context, we recently developed a new method of processing that combines simultaneously frontal ultrafiltration (FU) and ultrasound (US), to produce innovative orthotropic organizations on aqueous CNC suspensions (Pignon et al., 2024; Bosson et al., 2025a,b). A dedicated channel cell was developed to simultaneously generate a vertical acoustic force via a vibrating blade at the top and a transmembrane pressure force through a membrane at the bottom. Thanks to small angle X-ray scattering (SAXS), three specific

<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> Nantes Université, Oniris, CHU Nantes, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229, F-44000 Nantes, France</footnote>
orientations in three successive layers were revealed in this one-step FU-US process, mimicking the orthotropic organization of the collagen fibers inside the articular cartilage, with collagen fibers oriented horizontally in superficial zone, obliquely in intermediate zone and vertically in the deep zone (Bosson et al., 2025c; Camarero-Espinosa et al., 2016; Sophia Fox et al., 2009). Nevertheless, this organization is not preserved when the FU/US process is stopped. As revealed in previous measurements (Bosson et al., 2025a), in the absence of transmembrane pressure, the orthotropic structure had almost disappeared. With a view to potential applications in tissue engineering, this study investigates the effect of incorporating poly(ethylene glycol) diacrylate and collagen into CNC aqueous suspension on the preservation of this orthotropic structuring under same FU/US processing conditions. These additions aim respectively to enable future photocrosslinking of the aligned structure and to better mimic the biochemical composition of native cartilage tissue.

Collagen, the most abundant protein in the body, accounts for about 30% of total protein mass and serves as a primary structural element in various tissues, including cartilage (Bielajew et al., 2020; Dong & Lv, 2016). Its triple helix structure provides tensile strength and stability to the extracellular matrix, promoting cell adhesion and growth (Solouk et al., 2011; Somaiah et al., 2015). Additionally, the ability of collagen to self-assemble into liquid crystalline phases enhances its function as a scaffold material, mimicking the natural extracellular environment (Ghazanfari et al., 2016). However, the pH of the suspensions needs to be first equilibrated to an acidic condition. It has been shown that acetic acid (AA), by reducing the pH, effectively stabilizes collagen in solution, avoids collagen fibrillogenesis and facilitates its dispersion with other components in the suspension (Bak et al., 2018).

In order to study the ability of CNC suspensions, with a certain amount of PEGDA and with or without collagen, to be oriented in orthotropic organization under FU/US process, two successive approaches have been followed. First, we hypothesized that achieving liquid crystal-like behavior is essential for orienting the particles during ultrafiltration and ultrasound application (Bosson et al., 2025a; Pignon et al., 2021). In order to verify this assumption and define the experimental conditions (concentrations and ratio of CNC/PEGDA/collagen) for which this liquid crystal-like behavior is achieved, rheometric and polarized light microscopy measurements were performed. To further assess whether the orthotropic organization is preserved under different experimental conditions, a complementary approach based on original in situ dichroism measurements (Bosson et al., 2025b) was performed during the FU/US process. We also explored how filtration time and acoustic power influence the orientation of CNCs in these suspensions. This investigation provides critical insights into the interactions between PEGDA, collagen, and CNCs, ultimately informing future studies focused on developing stable composites for tissue engineering applications.

# 2. Materials and methods

## 2.1. Materials

CNC were obtained from the UMaine Development Center (University of Maine, USA) as a 10.6 wt% aqueous suspension. The dimensions of the CNCs were previously determined using SEM and image analysis. The CNCs were 127.4 ± 34 nm in length, and 10.9 ± 2.6 nm in width (Mandin et al., 2024). To disperse the CNC aggregates, the suspension was sonicated using a Branson Digital sonifier (250 W, Marshall Scientific, Hampton, NH). PEGDA (700 g.mol⁻¹) was sourced from Sigma-Aldrich (France). Collagen, which was acid-soluble and dry (from calf hides), was purchased from Symatech (France). 100 kDa polyethersulfone (PES) filtration membrane was purchased from Orélis Environnement (France).

## 2.2. Preparation of CNC suspensions

For CNC suspensions in water, the 10.6 wt% CNC stock was sonicated to break down aggregates, then diluted with deionized water to prepare final concentrations ranging from 5 to 10 wt% (noted as XCNC) (Bosson et al., 2025b; Mandin et al., 2024). CNC/PEGDA suspensions were prepared by mixing the sonicated CNC stock with PEGDA in an 80/20 weight ratio [CNC / PEGDA], resulting in CNC concentrations of 5, 6, and 8 wt% and noted as XCNC_20P, with X the CNC concentration (Metilli et al., 2025). For CNC/PEGDA/AA suspensions, CNC, PEGDA, and 0.5 M AA were added simultaneously, noted as XCNC_20P_0.5AA. Finally, CNC/PEGDA/AA/collagen suspensions were prepared by dissolving collagen powder in 0.02 M AA before combining it with the XCNC_20P_0.5AA suspensions. Collagen concentrations of 0.01, 0.1, and 0.5 mg/mL were tested, and the final suspensions were noted as XCNC_20P_0.5AA_YCol, with Y, the collagen concentration. All suspensions were stirred at room temperature for 2 hours to ensure homogenization.

## 2.3. Characterization of the suspensions

### 2.3.1. Rheometry

Flow curves were obtained using a DHR3 rheometer (TA Instruments) equipped with a smooth cone and plate geometry (angle: 0°: 59°: 42", diameter: 60 mm, truncation: 26 µm). The measurements were conducted at a constant temperature of 20°C. Shear rates were applied in the range of 0.01 to 1000 s⁻¹ to evaluate the rheological behavior of the suspensions, including XCNC, XCNC_20P, XCNC_20P_0.5AA and XCNC_20P_0.5AA_YCol, in order to assess their liquid crystal behavior.

### 2.3.2. Phase diagram

To create the phase diagram, the suspensions XCNC_20P_0.5AA_YCol, with X varying between 4 and 8 wt% and Y value of 0.01 and 0.1 mg/mL, were prepared and placed into 2 mm quartz cuvettes (Hellma Analytics, Germany), which were subsequently sealed with parafilm. The samples were left undisturbed for one week, allowing sufficient time for complete phase separation. For cholesteric phase characterization, photographs were taken using a camera (KY-F55B, JVC, Japan) while the cuvettes were positioned between crossed polarizers and illuminated with white light. To analyze the volume of the cholesteric phase, image processing was performed using ImageJ software. The percentage of the anisotropic phase for each concentration was determined by calculating the thickness of the cholesteric region relative to the total thickness of the suspension within the cuvette, following the methodology described in previous studies (Elazzouzi-Hafraoui et al., 2009; Mandin et al., 2024).

### 2.3.3. Polarized light microscopy (PLM)

The presence of fingerprints, indicative of cholesteric phase, was examined using a LEICA DM LM polarized light microscope (Leica, Germany), with magnification ranging from 5x to 50x. Suspensions were prepared by placing them on microscope slides one week prior to observation, utilizing the same samples as those analyzed with the rheometer. The cholesteric pitch *P* was determined by analyzing Fourier transform images of the fingerprint pattern, employing a custom image processing algorithm developed in Python (Metilli et al., 2025). 10 images were captured and analyzed for each sample, and the average pitch value was then determined.

## 2.4. In situ characterization during frontal ultrafiltration coupled to ultrasound

### 2.4.1. FU/US Cell description

A specialized channel-type filtration cell was developed at the "Laboratoire Rhéologie et Procédés" to allow the in situ observation of dynamic changes within CNC suspensions, possessing the same
characteristics as those used in previous studies (Jin et al., 2014, 2015; Pignon et al., 2024). Made of PMMA, the cell contains a parallelepiped channel that has dimensions of 4 mm in width, 8 mm in depth, and 100 mm in length. At the upper section of the cell, a titanium vibrating blade, measuring 3 mm in thickness and 100 mm in length, is installed at a height of z = 6400 µm above the ultrafiltration membrane surface. This blade connects to a sonotrode (Sodeva TDS, Méry, France) that consists of a 20 kHz piezoelectric transducer linked to a metal rod, generating ultrasonic vibrations. The CNC suspension is drawn from a reservoir using a syringe, ensuring the entire channel is filled. An inlet valve and an outlet valve are strategically positioned at the respective ends of the channel. A T-junction and a pressure sensor (FP 110 FGP Sensors & Instrument) are located between the outlet valve and the cell's outlet. Compressed air is fed into the upper section of the T-junction to maintain the integrity of the suspension's air circuit during the filling process, as already described in Bosson et al. (Bosson et al., 2025b). The permeate is subsequently collected in a designated container.

### 2.4.2. In situ dichroism

Dichroism is a laboratory experimental technique employed to determine particle orientation within a suspension. It offers valuable information on particle orientation at various length scales, ranging from nano to micro, and is more accessible than other methods such as SAXS, requiring less complex equipment. This method has previously been utilized to study the orientation dynamics of lyotropic colloidal suspensions of sepiolite clay subjected to extensional flow, providing the first experimental evidence of the "strong flow-weak flow" phenomenon in a three-dimensional system (Pignon et al., 2003). In our earlier research, we validated this technique for observing the orthotropic structure in a CNC suspension in water, thus justifying its application in the current study (Bosson et al., 2025b).

The optical setup for these experiments is illustrated in Fig. 1. It consists of a helium-neon laser that generates monochromatic light with a wavelength of 632 nm. The beam spot size is about 250 µm while the exposure time is 0.1 s. This light is directed through a straight prism and then passes through several components: a linear polarizer, a photoelastic modulator (PEM), a quarter-wave plate, the ultrafiltration cell, and a photodiode serving as a detector. The linear polarizer is oriented at 45° to the PEM, while the quarter-wave plate is aligned with the linear polarizer.

The measured intensity I can be expressed by the following equation:

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

where $I_{dc}$ represents the initial direct intensity passing through the sample after traversing the PEM and the quarter-wave plate, and $\alpha$ is the

<figure><img src="image_3.png" /><figcaption>Fig. 1. Optical set-up for the dichroism experiment.</figcaption></figure>

average fiber orientation angle relative to an arbitrary axis in the laboratory. The 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 $e$ 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).

### 2.4.3. Experimental protocol of the ultrafiltration process under in situ dichroism

To investigate the effects of filtration time and ultrasonic power on particle orientation, XCNC_20P and XCNC_20P_0.5AA_0.01Col suspensions were subjected to ultrafiltration coupled with ultrasound. For the effect of filtration time, suspensions XCNC_20P and XCNC_20P_0.5AA_0.01Col with X varying between 5 and 6 wt% were utilized. The filtration process was conducted for a duration of 2 hours under an acoustic power $P_a$ of 1.2 W.cm$^{-2}$ ($P_{US}$ = 20 %). During this period, vertical scans (z-scans) from the membrane surface to the vibrating blade were performed every 15 minutes, measuring the transmitted intensity through the sample in the filtration cell. To examine the effect of ultrasonic power, suspensions XCNC_20P with X varying between 5, 6, and 8 wt% were tested. The filtration was initiated for 1 hour under an acoustic power $P_a$ of 1.2 W.cm$^{-2}$. After this initial period, the acoustic power $P_a$ was incrementally increased (2.4, 3.6, 4.8 and 6 W.cm$^{-2}$) with scans conducted at each power level to monitor changes in particle orientation and structure. For all experimental conditions, an initial z-scan was performed before filtration started, and a final scan was conducted immediately after stopping the ultrasonic forces.

## 3. Results and discussion

### 3.1. Observation of colloidal behavior in CNC/PEGDA/Collagen suspensions

#### 3.1.1. Rheological analysis of CNC suspensions

CNC suspensions display a typical three-regime behavior under shear, characteristic of liquid crystalline systems, as shown by rheometric measurements (Fig. 2A). These regions reflect the progressive organization of cholesteric CNC phases influenced by electrostatic stabilization of colloidal particles, especially as shear rates increase (Derakhshandeh et al., 2013; Haywood et al., 2017; Gicquel et al., 2019; Kádár et al., 2021; Rosén et al., 2020; Sanchez-Botero et al., 2018; Shafiei-Sabet et al., 2012, 2014; Xu et al., 2017). The nature of these regimes is detailed below.

The first region (Region I) shows a rapid decline in viscosity as the shear rate rises, associated with the gradual orientation of cholesteric CNC domains. This initial ordering, where domains align their helical axes vertically, relieves internal stresses, facilitating flow. As described by Pignon et al. (Pignon et al., 2021), in situ small-angle light scattering (SALS) measurements under shear demonstrate how CNC cholesteric organizations progressively align in the vertical direction with increasing shear. This alignment contributes to a shear-thinning effect as internal resistance decreases. In the second region (Region II), viscosity stabilizes, forming a “plateau” sometimes referred to as the Newtonian region (Derakhshandeh et al., 2013; Onogi & Asada, 1980; Shafiei-Sabet et al., 2012, 2014; Xu et al., 2017). In this state, the CNCs reach a steady alignment within the flow, allowing continuous movement without further restructuring. The tactoids maintain their orientation under moderate shear, indicating a stable liquid crystalline arrangement without additional thinning. Pignon et al. (Pignon et al., 2021) also observed that during this phase, the cholesteric structure exhibits consistent flow behavior, as evidenced by the in situ SALS measurements that confirm the persistence of the aligned domains. The third region (Region III) marks a return to shear-thinning behavior as viscosity decreases with increasing shear. At these high shear rates, CNC domains undergo progressive fragmentation, disrupting the cholesteric structure
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** (A) Flow curve of XCNC suspensions in deionized water at different concentrations (5 to 10 wt%), revealing the three regions typical of liquid crystal. (B) Flow curve of XCNC_20P suspensions at different CNC concentrations (5, 6 and 8 wt%) with a constant PEGDA concentration (20 wt%). (C) Flow curve of XCNC_20P_0.5AA suspensions at different CNC concentrations (5, 6 and 8 wt%), 20 wt% of PEGDA and acetic acid at 0.5 M. (D) Flow curve of XCNC_20P_0.5AA_YCol at different CNC concentrations (5, 6 and 8 wt%), 20 wt% of PEGDA, acetic acid at 0.5 M and different concentration of collagen (0.01; 0.1 and 0.5 mg/mL). (E) Image of the inhomogeneous 8CNC_20P_0.5AA_0.5Col suspension.</figcaption></figure>

until complete dissociation and formation of a nematic-like arrangement of the individual CNCs (Pignon et al., 2021), leading to a common orientation direction of all CNCs and consequently a reduction of internal stresses leading to decreasing viscosity.

In summary, these three flow regions highlighted the structural transitions typical of cholesteric CNC suspension, from organized to fragmented states, under shear. These findings illustrate that this liquid crystal behavior has been observed in CNC suspensions at concentrations ranging from 5 to 10 wt%.

We hypothesized that the observed liquid crystal behavior in CNC suspensions is crucial for the effective orientation of CNCs during ultrafiltration coupled to ultrasound, obtained previously (Pignon et al., 2024; Bosson et al., 2025a–c). This behavior indicates that the CNCs maintain a specific interparticle distance characteristic of liquid crystals. Consequently, orienting one CNC can lead to the alignment of others, in the same direction, due to the cooperative interactions within the system. To evaluate how the concentration and the ratio of CNC/PEGDA/collagen affect this well-established liquid crystal-like behavior, a

series of rheometric measurements were performed. In the following, firstly the effect of the addition of PEGDA alone was studied and secondly the addition of collagen to CNC/PEGDA was studied.

### 3.1.2. Effect of PEGDA addition on the liquid crystal behavior

With the addition of 20 wt% PEGDA, the flow curves were obtained for different CNC concentrations (Fig. 2B). The three-regime behavior observed indicates that the CNC suspensions maintain a consistent liquid crystal behavior across the tested CNC concentrations of 5, 6, and 8 wt%, as previously studied in detail (Metilli et al., 2025). The flow curves for these suspensions demonstrate typical characteristics associated with cholesteric phases, confirming the liquid crystal behavior of the CNCs when combined with PEGDA (Metilli et al., 2025).

### 3.1.3. Effect of collagen addition on the liquid crystal behavior of CNC/PEGDA/Collagen suspensions

We next investigated the effect of collagen addition. To prevent collagen fibrillogenesis, which typically occurs at a range of pH 6-13 in
dilute solutions (Cooper, 1970), the suspension pH was adjusted to an acidic value of 2.8 using acetic acid. This acidic environment stabilizes collagen in solution, facilitating its homogeneous dispersion with CNC and PEGDA (Bak et al., 2018). A concentration of 0.5 M acetic acid was chosen, effectively preventing collagen fibrillogenesis and ensuring its stable dispersion with CNCs, as commonly reported in the literature (Li et al., 2015; Liu et al., 2022). Consequently, 0.5 M AA was added to maintain collagen in a soluble, non-fibrillar state within CNC/PEGDA suspensions, ensuring homogeneous dispersion and preserving their rheological properties. First, the effect of the decrease of pH was tested in the XCNC_20P_0.5AA suspensions (Fig. 2C), and the results showed that the typical three-regime liquid crystal behavior remained intact, with flow curves similar to those observed without acetic acid (XCNC_20P suspensions) (Fig. 2B). This observation suggests that the presence of acetic acid does not disrupt the cholesteric structure of CNCs.

When examining the CNC suspensions containing collagen at acidic pH (Fig. 2D), the three-regime typical of liquid crystal behavior was observed for CNC concentrations of 6 and 8 wt%, irrespective of the collagen concentration tested (0.01, 0.1 and 0.5 mg/mL). However, it is important to note that, for the suspension with the highest concentration of CNC (8 wt%) and collagen (0.5 mg/mL) in the presence of AA, the sample exhibited heterogeneity (i.e., presence of cloudy phases) and could not be tested (Fig. 2E). This observation highlights the potential challenges of maintaining a stable liquid crystal phase at higher concentrations of collagen and CNC. The inhomogeneity observed in this sample suggests that interactions between high concentrations of collagen and CNC may lead to aggregation, potentially disrupting the uniform cholesteric structure required for liquid crystal behavior.

For the lowest CNC concentration tested (5 wt%), the results were less straightforward. The typical three-regime liquid crystal behavior was not maintained in the suspensions with 0.5 mg/mL collagen (Fig. 2D), exhibiting more of a pseudoplastic behavior as shown in the supplementary data (Fig. S1). The region I of the flow curve was also absent in those containing lower collagen concentrations (0.01 and 0.1 mg/mL). This absence may be attributed to too low CNC concentration to establish a stable cholesteric phase under these conditions.

Overall, these findings emphasize the ability of CNC/PEGDA/

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Phase diagram of CNC in XCNC_20P_0.5AA_YCol suspensions (collagen concentration of 0.01 mg/mL in blue and 0.1 mg/mL in orange). In green, the phase diagram of XCNC_20P extracted from Metilli et al. (Metilli et al., 2025).</figcaption></figure>

Collagen suspensions to maintain the three-region behavior typical of liquid crystalline systems, with certain concentrations of CNC exhibiting stable cholesteric phases while others do not.

### 3.1.4. Phase diagram assessment of CNC systems

In order to complete the previous rheological analysis on how the concentration and ratio of CNC/PEGDA/collagen content affect the liquid crystal behavior of the studied system, a phase diagram analysis has been developed. This analysis was based on polarized light observations for samples in quartz cuvettes and in optical microscopy, both at rest. Indeed, a phase diagram illustrates the various structural states of CNC suspensions as a function of concentration, showing thresholds where specific behaviors such as cholesterics and isotropic arrangements are observed (Dong et al., 1996; Gicquel et al., 2019; Lagerwall et al., 2014; Lenfant et al., 2017; Revol et al., 1992; Schutz et al., 2015; Xu et al., 2020). This is especially relevant in this study for identifying conditions that lead CNCs to adopt specific organizations within a solution containing PEGDA and collagen. In our group, the phase diagrams for CNCs in deionized water and in the presence of PEGDA (20 wt%) were previously established (Mandin et al., 2024). For CNCs in deionized water, an isotropic phase appeared below 2.7 wt%, while a fully cholesteric phase occurred above 8 wt%, with a biphasic region between these concentrations (Mandin et al., 2024). In 20 wt% PEGDA, the biphasic range broadens, which is attributed to the adsorption of PEGDA on CNC surfaces, altering CNC morphology and reducing their chiral power, which affects phase behavior compared to pure CNC suspensions (Metilli et al., 2025).

In the same way, a phase diagram for XCNC_20P_0.5AA_YCol suspensions was obtained for two collagen concentrations, 0.01 and 0.1 mg/mL, and for CNC concentration ranging from 4 to 8 wt% (Fig. 3). The results indicate an extended biphasic region between 4 and 8 wt% CNC, with an increase in the cholesteric phase proportion at a given CNC concentration as collagen concentration increases (all the cross-polarized observations are shown in Fig. S2). While a slight cholesteric phase was previously observed at 4 wt% CNC in the absence of collagen, no such phase was detected at this concentration in the presence of collagen at acidic pH. This behavior points toward a specific influence of collagen on CNC interactions, likely stabilizing cholesteric ordering only at higher concentrations. Moreover, with a length of nearly 300 nm and a diameter of 1.5 nm (Vesentini et al., 2013), the size of the collagen is comparable to that of CNCs, which may lead to steric hindrance. This disruption may necessitate higher CNC concentrations to achieve stable cholesteric phases.

In comparing the rheological behavior and the phase diagram of CNC suspensions, distinct insights into the nature of cholesteric phases emerge. As illustrated in Fig. 2D and Fig. 3, cholesteric behavior is evident at 6 wt% CNC, with cholesteric phases of 88.6% and 89.1% for collagen concentrations of 0.01 and 0.1 mg/mL, respectively, and at 8 wt% CNC, where a cholesteric phase of 100% is observed for both collagen concentrations. In contrast, no cholesteric behavior is noted for 5 wt% CNC in the rheological data (Fig. 2D), a small cholesteric phase of 9.6% is still present in the phase diagram (Fig. 3), indicating a nuanced relationship between concentration and phase behavior.

### 3.1.5. Polarized light optical microscopy for cholesteric fingerprint observation

In order to have an additional analysis of the effect of PEGDA, acidic pH and collagen content on the liquid crystal behavior of the systems studied, polarized light microscopy observations were performed. The primary objective of these microscopic observations was to identify the presence of cholesteric phases within the CNC suspensions and their mixtures, which is visually indicated by the appearance of specific patterns known as fingerprints (Schütz et al., 2020; Voisin et al., 2023; Zhang et al., 2023). These structures are characteristic of the formation of cholesteric phases, where CNCs organize into helical arrangements, resulting in alternating light and dark bands under polarized light. These
observations were performed under several suspensions, namely XCNC, XCNC_20P, XCNC_20P_0.5AA, and XCNC_20P_0.5AA_YCol, with collagen at two distinct concentrations (0.01 mg/mL and 0.1 mg/mL). The formation of these patterns not only validates the existence of cholesteric phases but also allows for a comparison of the influence of various compounds and experimental conditions on the structural organization of the CNCs.

These findings are illustrated in Fig. 4, showing polarized light optical microscopy images of the different CNC suspensions across the various conditions. In each suspension, a distinct fingerprint pattern appears, confirming the formation of cholesteric phases and indicating a helical arrangement of CNCs that persists despite variations in polymer composition. This consistent fingerprint structure across all samples demonstrates that the addition of PEGDA and collagen does not disturb the cholesteric organization of CNCs, suggesting robust liquid crystal behavior across different experimental conditions.

To further analyze the cholesteric structure in our CNC suspensions, the cholesteric pitch size was estimated by measuring the distance between two bright lines in the fingerprint patterns. As described by others, in cholesteric liquid crystals, the spacing between two bright lines corresponds to half of the cholesteric pitch, allowing for an accurate estimation of the pitch size across samples (Voisin et al., 2023). In this study, measurements were taken between two fringes of similar texture in the fingerprint patterns (Fig. 5A). Fourier transform analysis was applied to images of the repeating fingerprint pattern using a custom image processing workflow in Python, allowing precise determination of pitch size by analyzing the spatial frequency of the fingerprint motifs, such as previously done (Metilli et al., 2025). A representative fingerprint pattern obtained for the 6CNC_20P suspension is shown in Fig. 5A. This example illustrates the cholesteric organization typically observed in these systems. The evolution of the cholesteric pitch size with CNC concentration is then generalized across various matrices (deionized water, 20 wt% PEGDA, and 20 wt% PEGDA with 0.5 M acetic acid and 0.01 mg/mL collagen), as presented in Fig. 5B.

The results in Fig. 5B show a clear trend in the evolution of pitch size as a function of CNC concentration. In water, the pitch size decreases with increasing CNC concentration, which is consistent with expected

behavior: as CNC concentration rises, particles are drawn closer together, resulting in a reduction in pitch size (Dong et al., 1996; Elazouzi-Hafraoui et al., 2009).

In presence of 20 wt% PEGDA, a similar trend is observed, where increasing the CNC concentration also leads to a decrease in pitch size. However, a significant reduction in pitch size is noted compared to CNCs in water. This difference can be attributed to the PEGDA content, which enhances interactions between CNCs, promoting tighter packing and stronger cholesteric ordering. A likely explanation is that PEGDA adsorbs onto the CNC surfaces, effectively increasing their thickness and thus their apparent volume fraction. As a consequence, the cholesteric helix may become tighter (Metilli et al., 2025). Specifically, the hydrophilic nature of CNCs promotes higher wettability on its surface, facilitating the adsorption of PEGDA, which enhances interfacial adhesion between the CNCs and PEGDA. This interaction is expected to strengthen the nanocomposite structure (Palaganas et al., 2017; Yang et al., 2013). Moreover, strong hydrogen bonding is established between the hydroxyl groups on CNCs and the carbonyl groups on PEGDA, as highlighted by Hou et al. (Hou et al., 2017), which increases attractive forces and contributes to the tighter packing of CNCs. As the concentration of CNC increases, these interactions become more pronounced, resulting in a denser arrangement of CNC.

A similar reduction in pitch size was observed when 0.01 mg/mL of collagen at acidic pH were added to the XCNC_20P suspensions (Fig. 5B). It seems that the decrease of pH, in presence of collagen, enhances the interactions between the CNC particles. Additionally, collagen can also contribute to the intermolecular interactions within the suspension. This combination likely creates an environment that promotes closer interactions between CNC, leading to a similar reduction in cholesteric pitch size. These results suggest that PEGDA already induces a maximum effect on CNC organization, such that the additional presence of collagen in acidic pH does not further influence the pitch.

To summarize, the rheological analysis indicated that all suspensions, except for the 5CNC_20P_0.5AA_YCol suspension with collagen at concentrations of 0.01, 0.1, and 0.5 mg/mL, exhibited the typical three regime liquid crystal behavior across the tested shear rate range (0.01 to 1000 s⁻¹). Microscopy observations confirmed the liquid crystal behavior, thanks to the presence of cholesteric structures through the

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Polarized light optical microscope observation (x20) of CNC suspensions (5, 6 and 8 wt%), in water, or in the presence of PEGDA (20 wt%), acetic acid (0.5 M) and collagen (0.01 and 0.1 mg/mL). Scale bar: 50 µm.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** (A) Polarized light optical microscopic image of a 6CNC_20P suspension showing characteristic fingerprint patterns. (B) Evolution of fingerprint size measured from microscope images as a function of CNC concentration in different matrices: water, 20 wt% PEGDA, and 20 wt% PEGDA with 0.5 M acetic acid and 0.01 mg/mL collagen.</figcaption></figure>

appearance of characteristic fingerprints. Based on these findings, we then studied the effect of combined FU and US on the structure organization of all these suspensions. We expected that, in certain conditions, an orthotropic organization could be achieved. We also hypothesized that there could be a correlation between the three typical regions of liquid crystal behavior and the ability to obtain this orthotropic organization.

## 3.2. Observation of orthotropic structure under ultrafiltration and ultrasound

In the aim to verify the ability of the system to obtain the orthotropic organization, two types of investigation were performed to study the conditions under which this structure appears. Firstly, the impact of filtration time was investigated on suspensions XCNC_20P and XCNC_20P_0.5AA_0.01Col, with CNC concentration of 5 and 6 wt%. Secondly, the impact of the acoustic power was examined on suspensions XCNC_20P with CNC concentration of 5, 6 and 8 wt%.

### 3.2.1. Investigating the impact of filtration time on orthotropic structure

Dichroism has previously been employed to explore the orientation changes of CNCs in aqueous solutions during FU combined with US (Bosson et al., 2025b) and allowed for the capture of real-time variations in CNCs orientation and transmitted intensity during the filtration process. In our current study, this methodology was extended to investigate the organization of CNCs in the presence of PEGDA and collagen in acidic pH, allowing to analyze their behavior in a more complex medium. To investigate the effect of filtration time, suspensions were subjected to a filtration process lasting 2 hours under a constant acoustic power $P_a$ of 1.2 W.cm$^{-2}$. Dichroism measurements were conducted at regular 15-minute intervals using z-scans within the filtration cell.

For the XCNC_20P suspensions (Fig. 6A and Fig. 6B), dichroism showed a change of the orientation of the particles during the ultrafiltration process, from 90° to nearly 190° at around 3 and 4 mm from the membrane surface, respectively for the 5 and 6 wt% of CNC. Initially, prior to the ultrasound-assisted filtration, the particle orientation angle (red curve) shows a consistent angle of approximately 90° across the height of the filtration cell. This initial state is indicative of the shear-induced alignment of CNCs that occurs during the preparation and the pumping of the suspension into the filtration cell. The data reveal a clear transition in the orientation of the CNCs during ultrafiltration combined to ultrasound. Firstly, the orientation angles oscillate around 90°, indicating a predominantly horizontal alignment of the CNCs, parallel to the

membrane surface. However, beyond approximately 3 to 4 mm from the membrane surface, a significant shift occurs. The orientation angles progressively transition to around 180°, suggesting a distinct change in orientation: the CNCs exhibit a horizontal orientation close to the membrane and shift to a vertical orientation further away, approaching the vibrating blade. The orientation of CNCs parallel to the membrane near the membrane surface can be attributed to the pressure effect exerted during ultrafiltration, which forces the particles to orient in this manner (Mandin et al., 2024; Rey et al., 2019). Conversely, the vertical orientation observed near the vibrating blade is likely influenced by the acoustic streaming generated by the ultrasound, which actively reorients the CNCs in the suspension (Pignon et al., 2024; Bosson et al., 2025a,b).

For the XCNC_20P_0.5AA_0.01Col suspensions with 5 and 6 wt% CNC represented in Fig. 6C and Fig. 6D respectively, distinct behaviors are observed concerning particle orientation during the ultrafiltration process combined to ultrasound. In Fig. 6C, representing the 5CNC_20P_0.5AA_0.01Col suspensions, shows a different behavior. Throughout the 2 hours of filtration, no significant change in orientation is detected, with the angles remaining consistently around 90° across the height of the ultrafiltration cell. Especially in the region close to the vibrating blade, where ultrasonic forces are dominant, no change in the orientation of the CNCs could be detected. In the same time, the rheological study (Fig. 2) indicates that the 5CNC_20P_0.5AA_0.01Col suspension did not display any liquid crystalline properties within the tested shear rate range (0.01 to 1000 s$^{-1}$). These observations align with our initial hypothesis that liquid crystalline behavior is crucial for achieving orthotropic orientation of CNCs during FU-US processing.

In contrast, in Fig. 6D, with a CNC concentration of 6 wt%, the same orientation as seen previously is observed. Near the membrane, the CNCs exhibit a horizontal alignment, while a vertical orientation is observed at distances of 3 to 4 mm above the membrane surface, particularly close to the vibrating blade. However, as the filtration time increases, the angles near the membrane fluctuates (Fig. 6D), suggesting a higher concentration of cholesteric structures forming in this region. It is confirmed by Fig. S3 showing the presence of cholesteric structures that are not well oriented near the membrane, which appears to justify the less defined angles in this area.

In conclusion, the investigation has confirmed our hypothesis that liquid crystalline behavior is necessary to achieve orthotropic orientation of CNC. The results also demonstrate that a filtration time of 60 minutes is enough to induce the formation of a well-defined orthotropic structure. Therefore, this intermediate time of 60 minutes will be
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Effect of filtration time. Particle orientation angle measured by dichroism for XCNC_20P suspensions at different CNC concentrations: 5 wt% (A,C) and 6 wt% (B, D), for different filtration times (from 0 to 120 min) with a constant acoustic power $P_a = 1.2 \text{ W.cm}^{-2}$. A and B correspond to XCNC_20P suspensions, while C and D correspond to XCNC_20P_0.5AA_0.01Col suspensions. Schematic representations of CNCs orientation have been added to each figure.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Effect of US power on particle orientation. Particle orientation angle measured by dichroism for XCNC_20P suspension with (A) 5 wt% CNC, (B) 6 wt% CNC and (C) 8 wt% CNC at varying acoustic power levels. Measurements were taken before filtration ($t_f = 0$ min) and after 60 min of filtration, with acoustic power ranging from 1.2 to 6 W.cm$^{-2}$.</figcaption></figure>
utilized in subsequent experiments to explore the effects of acoustic power on CNCs orientation. Moreover, since the addition of macromolecules, such as collagen, does not disturb this orthotropic structure, the rest of this study will focus only on CNC/PEGDA suspensions, without those macromolecules.

### 3.2.2. Influence of acoustic power in orthotropic structure formation on XCNC_20P suspensions

Dichroism measurements were then used to monitor the effects of incrementally increased acoustic power on CNCs structural organization within the FU-US cell. To investigate the effect of acoustic power on CNCs orthotropic orientation, XCNC_20P suspensions at CNC concentrations of 5, 6, and 8 wt% were initially filtered for one hour under the lowest acoustic power ($P_a$ = 1.2 W.cm$^{-2}$). Following this initial period, the acoustic power was incrementally increased to 2.4, 3.6, 4.8, and 6 W.cm$^{-2}$. For each power level, a vertical z-scan was conducted to measure both transmitted light intensity and CNCs orientation angle (Fig. 7). The red curve represents the initial CNCs orientation before filtration, with an angle around 90°, corresponding to the orientation set by shear forces during the initial suspension setup. Regardless of the acoustic power applied, a change in particle orientation was observed, with the CNCs aligning horizontally near the membrane and vertically near the vibrating blade, indicating the formation of the orthotropic structure. A slight difference in the thickness of the oriented layers is noted depending on the acoustic power applied. Specifically, as the acoustic power increases, the layer oriented vertically by the ultrasound also increases in thickness. Although out of scope of this study, precise layer thickness measurement would be an interesting aspect to explore in future work, for example using SAXS as done in our previous study on orthotropic CNCs structuring in water (Bosson et al., 2025b).

In summary, varying ultrasonic power effectively influences CNCs orientation in suspensions containing PEGDA, consistently producing the orthotropic structure across all power levels. Higher powers increase the thickness of the vertically oriented layer near the ultrasonic blade. Our results demonstrated that precise control of acoustic power is essential for achieving optimal CNCs structural organization within complex biomaterial systems.

## 4. Conclusions

This study explored the effect of adding macromolecules on the liquid crystal-like behavior and orthotropic organization of CNCs. Notably, we observed liquid crystal behavior in the CNC/PEGDA suspensions, which we proved to be essential for achieving the desired orientation of CNCs under FU and US. The application of the dichroism method allowed us to corroborate this by examining CNC orientation during FU coupled with US. The orthotropic structure was successfully achieved under specific formulation conditions: i) without collagen, using 20 wt% PEGDA and 5 to 8 wt% CNC; and ii) with collagen at acidic pH, using 20 wt% PEGDA, 6 to 8 wt% CNC, and 0.01 to 0.1 mg/mL collagen. These results highlight that, under these conditions, orthotropic orientation is maintained, with acoustic power and filtration time playing a key role in preserving the orthotropic arrangement. These findings pave the way for future research, particularly into fixing the CNC orthotropic organization by photocrosslinking, and into exploring the biological properties of the nanocomposites. The successful formation of an orthotropic structure, reminiscent of the anisotropic organization of native articular cartilage, suggests the potential to develop scaffolds that could support cell growth and contribute to cartilage tissue engineering strategies.

### CRediT authorship contribution statement

F. Bosson: Writing – review & editing, Writing – original draft, Investigation, Formal analysis. A. Laviron: Investigation, Formal analysis. M. Karrouch: Methodology, Conceptualization. L. Metilli: Writing

– review & editing, Methodology. N. Hengl: Writing – review & editing, Supervision. V. Delplace: Writing – review & editing, Supervision. F. Pignon: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, 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 thank Didier Blésès, Frédéric Hugenell and Eric Faivre (Laboratoire Rhéologie et Procédés) for their technical assistance. 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 Glyco@Alps programme (Investissements d'Avenir - grant agreement #ANR-15-IDEX-02).

### Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.carpta.2025.101015.

### Data availability

Data will be made available on request.

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