# Designing Biomaterials with Oriented Organic Nanocrystals for Tissue Engineering Applications

Fanny Bosson, Nicolas Hengl, Baptiste Charbonnier, Jérôme Guicheux, Frédéric Pignon, Vianney Delplace

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## To cite this version:

Fanny Bosson, Nicolas Hengl, Baptiste Charbonnier, Jérôme Guicheux, Frédéric Pignon, et al. Designing Biomaterials with Oriented Organic Nanocrystals for Tissue Engineering Applications. Acta Biomaterialia, 2025, 208, pp.1-19. 10.1016/j.actbio.2025.10.021. hal-05323469

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## Review article

# Designing biomaterials with oriented organic nanocrystals for tissue engineering applications

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Fanny Bosson<sup>a, ID</sup>, Nicolas Hengl<sup>a</sup>, Baptiste Charbonnier<sup>b</sup>, Jérôme Guicheux<sup>b</sup>, Frédéric Pignon<sup>a, ID</sup>, Vianney Delplace<sup>b,* ID</sup>

## ARTICLE INFO

**Keywords:**

Nanocrystals

Biomaterials

Hydrogels

Manufacturing techniques

Tissue engineering

## ABSTRACT

The extracellular matrix (ECM) has a specific architecture that defines tissue mechanics and helps to guide cell functions. In tissue engineering, it is crucial to be able to mimic the architecture and mechanics of the ECM with biomaterials. However, mimicking the architecture and mechanics of tissues that contain ECM elements oriented at the nanoscale, such as articular cartilage, tendon, intervertebral disc, and cornea, remains challenging. To succeed, an emerging strategy is to orient nanocrystals within materials by using manufacturing techniques such as extrusion printing and magnetic induction. This strategy is increasingly used in tissue engineering where it can help to produce mechanically reinforced biomaterial scaffolds and spatially organize cells. This review introduces techniques that allow for orienting organic nanocrystals within materials and discusses their potential and limitations for tissue engineering applications. First, it presents an overview of the classes of organic nanocrystals used in tissue engineering, before summarizing the state of the art in the processing of oriented nanocrystals. Then, the use of materials containing oriented nanocrystals is discussed for two major applications, i.e., mechanical reinforcement and cell alignment. Finally, new avenues of research are proposed to advance this emerging class of materials toward biomedical applications.

**Statement of significance:** In tissue engineering, one major challenge is to mimic the architecture of the cell microenvironment down to the nanoscale. To succeed, one strategy is to use fabrication techniques that allow the orientation of nanocrystals within materials. This review presents the state of the art in the design of biomaterials with oriented organic nanocrystals and discusses their potential in tissue engineering.

## 1. Introduction

Nanoparticles, which are objects possessing dimensions in the range of one to a few hundreds of nanometers, can exhibit either an amorphous structure or a crystalline structure, which is an ordered arrangement of atoms [1]. When nanoparticles possess a crystalline structure, they are specifically referred to as nanocrystals. Anisotropy, which refers to variations in the physical properties of a material in three dimensions, plays a crucial role in the properties of materials built with nanocrystals. Indeed, the properties of nanocrystal-based materials depend on the orientation and spatial organization of their anisometric constituent particles. Due to their nanometric dimensions and anisometric form, nanocrystals and nanocrystal-based materials have become essential components in many scientific and technological fields [2], including electronics [3], photovoltaics [4], but also in biomedical fields such as

drug delivery [5,6] and thermal therapy [7,8]. More recently, nano-crystals have attracted increasing interest for tissue engineering applications, where they can help to design materials that mimic the mechanical and hierarchical structures of biological tissues.

Many biological tissues, including articular cartilage [9], tendon [10], muscle [11], intervertebral disc [12], and bone [13] present an anisotropic, hierarchical structure essential to their biomechanical and biological functions. Engineering materials that mimic such tissues requires recapitulating their anisotropic structures down to the nanoscale. To succeed, one of the main approaches consists in embedding oriented nanocrystals in the designed materials. However, controlling nano-crystal orientation within materials is challenging due to the difficulty of reproducibly and uniformly manipulating nanoscale structures, which are influenced by colloidal forces that can promote aggregation or disrupt alignment. To overcome this engineering challenge, a range of

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

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

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: vianney.delplace@univ-nantes.fr (V. Delplace).</footnote>
manufacturing techniques have been proposed, from 3D printing and calendering to unidirectional freezing, with promising results. Yet, these approaches have rarely been applied to tissue engineering, largely due to the complexity of their implementation, high costs, and challenges in scaling up for large-scale integration [14].

In this review article, we aim to bridge existing techniques and processing methods for orienting organic nanocrystals within materials and their potential use in tissue engineering. First, we will introduce the various types of organic nanocrystals commonly used in tissue engineering, detailing their sources, specific properties, and synthesis methods. Then, we will provide a comprehensive overview of the processing methods reported to date that enable the orientation of organic nanocrystals within materials. In a third section, we will summarize the state of the art in the design of oriented organic nanocrystals-containing materials for tissue engineering applications, which include the mechanical reinforcement of hydrogels and the promotion of cell alignment. Finally, we will discuss current challenges and innovations in the field, as well as future perspectives for enhancing the design, functionality, and clinical applications of engineered tissues.

## 2. Organic nanocrystals in tissue engineering: sources, processing, and properties

Nanocrystals can be synthesized from either inorganic or organic raw materials (Table 1). Each type of nanocrystal is associated with a specific synthesis method. Their physicochemical properties depend on their composition, shape and size, which may drastically differ from their amorphous homologues [15]. While metal-based nanocrystals show potential for tissue engineering applications, their potential cytotoxicity and immunogenic responses limit their applicability in biological environments [16–18]. However, despite these limitations, ongoing research aims to overcome these challenges by modifying the surface of metal-based nanocrystals or integrating them into biomaterial matrices to enhance their biocompatibility and efficacy in tissue engineering applications [19,20]. Even though these recent advances have been made, we will focus exclusively on organic nanocrystals that are utilized in tissue engineering.

<table><caption>**Table 1**

Characteristics of organic nanocrystals used in tissue engineering.</caption><thead><tr><th>Molecule</th><th>Source</th><th>Process</th><th>Length (in nm)</th><th>Diameter (in nm)</th><th>Aspect ratio (length/diam. ratio)</th></tr></thead><tbody><tr><td rowspan="5">Cellulose</td><td rowspan="5">Cotton</td><td>Acid hydrolysis</td><td>17 [21] – 200 [22]</td><td>4 [23] – 16.8 [21]</td><td>≈ 50*</td></tr><tr><td>Tunicates</td><td>2200 [24,25]</td><td>25 [24, 25]</td><td>80 [24, 25]</td></tr><tr><td>Wood (abaca. palm. spruce. bleached pulp)</td><td>44 [26] – 400 [27]</td><td>2.3 [26] – 20 [27]</td><td>22 [28, 29] – 94 [30]</td></tr><tr><td>Bacteria (Acetobacter xylinum)</td><td>237 [31]</td><td>–</td><td>–</td></tr><tr><td>Lobster exoskeleton</td><td>200 [32,33]</td><td>30 [32, 33]</td><td>≈ 7*</td></tr><tr><td rowspan="3">Chitin</td><td rowspan="3">Crab shells</td><td>Acid hydrolysis</td><td>100 – 500 [34]</td><td>15 – 30 [34]</td><td>3 – 16 [34]</td></tr><tr><td rowspan="2">Shrimp shells</td><td>300 [35]</td><td>20 [35]</td><td>15 [35]</td></tr><tr><td>–</td><td>–</td><td>–</td></tr><tr><td>Kartogenin</td><td>–</td><td>Wet milling process</td><td>320 [36]</td><td>–</td><td>–</td></tr><tr><td>Starch</td><td>Corn</td><td>Acid hydrolysis</td><td>58 [37]</td><td>–</td><td>–</td></tr></tbody></table>

### 2.1. Sources and synthesis methods

Organic nanocrystals are produced from natural polymers extracted from plants (e.g., cellulose [38], starch [39]) or from animals such as crustacean waste (e.g., chitin [40]) or tunicates (e.g., cellulose [38]). These diverse sources provide an opportunity to utilize renewable and biodegradable materials for nanocrystal fabrication.

Organic nanocrystals are mainly produced by acid hydrolysis. Acid hydrolysis consists in dissolving the amorphous chains of polysaccharides, using either sulfuric acid ($H_2SO_4$) or hydrochloric acid (HCl), thereby freeing crystalline domains [39]. The obtained nanocrystals can then be sonicated to be individualized and separated [41]. As with inorganic nanocrystals, the processing parameters (i.e., raw material, type of acid, acid concentration, temperature, and duration) can be adjusted to tune the size and morphology of the nanocrystals, offering flexibility in the design of these structures [39,42]. In addition to acid hydrolysis, the wet milling process has also been used to recrystallize kartogenin purchased as a partially crystalline powder [36, 43]. This process involves soaking raw materials in water to soften the grain and facilitate separation of its various components, which enables the production of high-quality nanocrystals.

### 2.2. Properties

Cellulose and chitin are the most commonly used organic compounds for the production of nanocrystals [42]. These two polymers form nanocrystals with similar mechanical properties, with a high Young's modulus (110–220 GPa [38] and ≈150 GPa [35], respectively) and a high tensile strength (7.5 to 7.7 GPa for cellulose nanocrystals [CNCs] [38]). These two types of nanocrystals commonly exhibit a large specific surface area and a high aspect ratio (from 3 to 16 for chitin nanocrystals [34], and from 10 to 30 for CNCs [38]). In contrast, starch nanocrystals have weaker mechanical properties than cellulose and chitin nanocrystals (Young's modulus of 1-20 MPa, and tensile strength of around 3 MPa) [44]. Starch nanocrystals typically form as platelets, i.e., flat, plate-like structures, with lengths ranging from 20 to 200 nm, widths from 10 to 30 nm, and thickness from 5 to 10 nm [39]. Cellulose and chitin nanocrystals can organize into cholesteric phases, characterized by a helical structure where the crystals gradually align, creating unique optical patterns [45–47]. This specific arrangement is mainly attributed to the chirality of the nanocrystals and the short and long range of colloidal interactions; but the origin of the cholesteric organization is still under debate.

Organic nanocrystals are gaining increasing attention for their potential applications in the biomedical fields [48]. They can be utilized as additives in substrates for cell culture [49], promoting cell attachment and growth [50,51]. Their cytocompatibility and ability to encapsulate therapeutic agents make them attractive for the design of drug delivery systems [52]. Recent studies also show a growing interest in nanocrystal-conjugated drugs, highlighting their potential for enhancing drug solubility and bioavailability [53–55]. Additionally, other organic compounds have been used to produce nanocrystals with specific properties. For example, perylene nanocrystals exhibit optical properties such as optical waveguiding behaviors [56] that depend on their size [57]. More recently, it was demonstrated that kartogenin, which is known to promote the chondrogenic differentiation of human mesenchymal stem cells, forms nanocrystals, with potential applications in the treatment of joint diseases (e.g., osteochondral defects, osteoarthritis) [58].

## 3. Processing materials with oriented nanocrystals

Nanocrystals can be added to a material by simply mixing them with the material's precursor (i.e., monomer or polymer solutions) before formation by polymerization or cross-linking. However, these methods do not spontaneously orient nanocrystals, which have, by default, an

<footnote>* Calculated from the diameters and lengths provided in the articles.</footnote>
isotropic orientation within materials. Thus, the use of additional pro-
cessing techniques is necessary to orient anisometric nanocrystals within
material scaffolds. Particle size and aspect ratio influence orientation
outcomes by affecting how nanocrystals respond to external fields or
processing conditions. These techniques can be based on shear, electric
and magnetic fields, and unidirectional freezing (Figs. 1 and 2). Before
discussing these processing techniques, we will first present the char-
acterization methods that allow for the precise quantification of nano-
crystal alignment, providing a deeper understanding of their
orientation. In this context, orientation refers here specifically to the
preferential spatial arrangement of individual nanocrystals within the
material. While such nanoscale alignment can influence higher levels of
structural organization, this review focuses primarily on the nanoscale
level.

3.1. Characterization of nanocrystal orientation

Various characterization techniques have been developed to quantify
the alignment of nanocrystals within materials (Table 2). These tech-
niques offer insights into the orientation of nanocrystal at different
scales. In particular, scattering techniques are powerful tools for
analyzing the orientation of nanostructures across multiple scales, from
atomic arrangements to mesoscopic and micrometric structures. These
methods rely on the interaction of electromagnetic waves (X-rays or
light) with the material, producing scattering patterns that contain in-
formation about the structural anisotropy and alignment of particles.
Three main scattering techniques, which provide complementary in-
formation, can be used: wide-angle X-ray scattering (WAXS); scattering
methods like small-angle x-ray (SAXS); and small-angle light scattering
(SALS).

WAXS is an effective method for analyzing the crystallographic
orientation of nanocrystals at an atomic scale, typically focusing on the
arrangement of atoms within individual crystals [59]. To achieve this,
one of the key data processing techniques is azimuthal analysis, which
involves measuring the variation in diffraction intensity as a function of
the azimuthal angle around the direction of the crystal axis. The in-
tensity of diffraction peaks obtained by WAXS can be analyzed at

different azimuthal angles to assess the alignment of crystals within
materials. For example, in oriented polymer films, such as blends of
thermotropic liquid crystalline polymers and polypropylene, a WAXS
analysis allowed the quantification of the orientation of crystals relative
to the drawing direction [60]. Similarly, in anisotropic powders made of
nanometric crystals, a 2D-WAXS image can provide information on the
orientation distribution of the crystals [61]. SAXS complements WAXS
by targeting larger structures at a mesoscopic scale (i.e., a few to hun-
dreds of nanometers). Like WAXS, SAXS provides radial and azimuthal
averages of the scattering intensities, which are used to assess the
orientation of crystalline structures [62]. This approach is particularly
suited to analyzing systems such as gels, oriented films, or anisotropic
powders. Finally, SALS is a complementary technique to SAXS and
WAXS that is used to analyze the orientation of nanocrystals at meso-
scopic and micrometric scales. Using polarized light instead of X-rays,
SALS produces scattering patterns that reveal the orientation distribu-
tion of larger structures, such as oriented films or gels. SALS allows for
the rapid, non-destructive analysis of anisotropy over large scale (i.e.,
from hundreds of nanometers to hundreds of micrometers) of the ma-
terial [63,64]. To quantify anisotropy and particle orientation from
WAXS, SAXS, and SALS measurements, the order parameter and Prin-
cipal Component Analysis (PCA) can be calculated [65]. The order
parameter, which is derived from the orientation distribution of parti-
cles, quantifies the degree of alignment relative to a director axis and
reflects both the anisotropy and alignment in materials such as gels and
oriented films. PCA offers a complementary approach by analyzing
multidimensional SAXS data clouds. It identifies the principal directions
of alignment by maximizing the variance of the projected data by
solving an optimization problem. This method provides a quantitative
measure of orientation, with values ranging from 0 (for isotropic sus-
pensions) to 1 (for completely aligned systems).

Beyond scattering techniques, AFM is a particularly powerful
method for characterizing the alignment and organization of nano-
crystals at the microscopic scale [66,67]. With its sub-nanometer reso-
lution, AFM allows direct visualization of the morphology of
nanocrystals, including their orientation, size, and distribution in com-
plex structures. Typically, AFM can be used to quantify the organization

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Schematic of the orientation of nanocrystals by different manufacturing methods. (a) extrusion-based 3D printing; (b) shear flow and ultrafiltration; (c) calendering; and (d) unidirectional freezing.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Schematic of the orientation of nanocrystals by different manufacturing methods. (a) induction by an electric field; (b) electrospinning; (c) electro-compaction; and (d) induction by a magnetic field.</figcaption></figure>

of CNCs by distinguishing isotropic phases from liquid crystalline phases and characterizing the helical structures of aligned CNCs in certain conditions.

Finally, at the molecular scale, polarized Raman spectroscopy is a key tool for detecting and quantifying local anisotropy [68]. It probes the alignment of chemical bonds in nanocrystals by studying characteristic bands, such as the one at $1095 \text{ cm}^{-1}$, which is associated with the C—O stretching of cellulose chains. As demonstrated by Rusli et al. [69], this method provides direct quantification of stress transfer under axial deformation, while distinguishing isotropic and anisotropic domains in composites. Additionally, it is sensitive to environmental conditions, such as humidity or temperature, allowing for the assessment of their impact on interactions within the network.

In conclusion, characterization methods for the orientation of nanocrystals, such as WAXS, SAXS, SALS, AFM, and Raman spectroscopy, provide a comprehensive, multi-scale analysis of crystal alignment. WAXS, SAXS and SALS give an overall view of the internal and global organization, while AFM and Raman spectroscopy explore alignment at the molecular and surface levels. The combination of these techniques allows for precise quantification of anisotropy and

orientation, which is essential for establishing links between structure and properties in nanocrystal-based materials. Of note, other characterization methods can be used to observe qualitatively nanocrystals orientation, such as polarized optical microscopy and electron microscopy.

## 3.2. Nanocrystal orientation using shear-flow processes

The orientation of organic nanocrystals by shear can be achieved through several methods and processes including extrusion-based 3D printing, filtration, calendering, and unidirectional freezing, each providing distinct mechanisms for controlling the orientation and organization of nanocrystals within materials.

### 3.2.1. Extrusion-based 3D printing

Extrusion-based 3D printing is an additive manufacturing technique that allows for the creation of complex structures by adding materials layer by layer. This process has gained immense popularity across various fields, including biomedical applications, aerospace, and consumer goods, due to its ability to produce customized and intricate

<table><caption>**Table 2**

Characterization methods for nanocrystal orientation.</caption><thead><tr><td>Method</td><td>Scale of analysis</td><td>Parameters Studied</td><td>Obtained information</td><td>Advantages</td><td>Limitations</td></tr></thead><tbody><tr><td>WAXS<sup>a</sup></td><td>Atomic</td><td>Crystallographic orientation</td><td>Crystal orientation and phase distribution</td><td>High crystallographic resolution</td><td>High-quality X-ray source and specialized equipment</td></tr><tr><td>SAXS<sup>b</sup></td><td>Mesoscopic</td><td>Nanocrystal alignment and anisotropy</td><td>Nanocrystal orientation and mesoscale anisotropy</td><td>Mesoscale alignment information in complex materials</td><td>Less precise for atomic-level details</td></tr><tr><td>SALS<sup>c</sup></td><td>Mesoscopic to microscale</td><td>Crystallite orientation distribution and anisotropy</td><td>Orientation distribution and microscopic anisotropy</td><td>Non-destructive, can probe larger sample areas quickly</td><td>Lower resolution than SAXS/ WAXS, scattering from larger structures</td></tr><tr><td>AFM<sup>d</sup></td><td>Surface to nanoscale</td><td>Nanocrystal orientation, size and morphology</td><td>Direct visualization of morphology, orientation, and distribution</td><td>High spatial resolution, direct visualization</td><td>Surface-limited characterization, slow analysis</td></tr><tr><td>Raman spectroscopy</td><td>Molecular</td><td>Local anisotropy, chemical alignment</td><td>Molecular alignment, stress transfer, environmental sensitivity</td><td>Sensitive to molecular alignment and stress in nanocrystals</td><td>Sample preparation sensitivity</td></tr></tbody></table>

<footnote><sup>a</sup> Wide-Angle X-ray Scattering, <sup>b</sup>Small-Angle X-ray Scattering, <sup>c</sup>Small-Angle Light Scattering, <sup>d</sup>Atomic Force Microscopy.</footnote>
designs that traditional manufacturing methods often cannot achieve [70]. One key aspect that enhances the functionality and performance of 3D-printed structures is the control of the alignment of nanoparticles during the printing process.

It is well known that anisotropic particles align during extrusion [71–73]. Under the effect of induced shear and flow velocity, nano-particles tend to align themselves parallel to the movement of the printhead, leading to a preferential orientation along the printed filament. This physical principle applies to all nanoparticles, including nanocrystals. The level of nanocrystals alignment achieved through extrusion-based 3D printing can vary considerably depending on the rheological properties of the ink and the type of flow imposed during the printing process. Interestingly, it has been shown that the flow-induced alignment of nanocrystals in highly concentrated inks is only possible if the applied stress exceeds the yield strength of the suspension [74]. The alignment was characterized using optical imaging techniques to capture the time-dependent orientation of CNCs under controlled shear in a rheometer. With the yield strength exceeded, the time needed for aligning anisotropic particles under shear can be quantified through a scaling relationship, which considers the applied shear rate and particle interactions at different volume fractions. At low applied shear rates, there is typically a linear relationship between alignment time and the inverse of the shear rate, offering a reliable approximation of the alignment process (readers can refer to the article of Hausmann et al. [74] for equation details). However, deviations from this linear pattern occur at higher shear rates, where steric and hydrodynamic particle interactions are presumed to have a more pronounced impact.

Building on this concept, another study investigated extrusion-based 3D printing for the production of hydrogels containing oriented CNCs, with potential applications in tissue regeneration (e.g., bone or cartilage) [75]. Using WAXS, the authors measured the alignment of CNCs by analyzing the shape of the azimuthal peak and applying an original calculation method (see article for details). It was confirmed that CNCs were preferably oriented in the printing direction, with a degree of orientation between 61 and 76%. The anisotropy of the printed scaffolds was also demonstrated by optical measurements (i.e., birefringence), confirming the shear-induced orientation of the CNCs during extrusion. SEM images completed this observation and confirmed the orientation of CNCs in the printing direction.

In summary, nanocrystals alignment by extrusion-based 3D printing is a promising method that offers the possibility for creating complex structure with high precision and control over nanocrystal orientation. However, this technology presents challenges, such as low manufacturing resolution, slow 3D printing speeds, and limited material choices [73]. For the effective alignment of nanocrystals by extrusion-based 3D printing, it is crucial to meet certain conditions: the nanocrystals must have an anisotropic shape, the applied shear stress during extrusion should exceed the yield strength of the suspension, and the rheological properties of the ink should be favorable for controlled flow. Additionally, the shear rate must be carefully optimized, as too high or too low shear rates can affect the alignment quality. In the future, a better understanding of these aspects will be crucial to making nanocrystal orientation by extrusion-based 3D printing viable on a large scale.

### 3.2.2. Filtration-based processes

Filtration is a separation process commonly used in various industries, including food, water and biomedical applications, that employs liquid/liquid filtration to filter particles or solutes from liquids, including viruses and bacteria. This technique involves applying a difference of pressure to a suspension through a porous membrane or filter, which selectively blocks particles based on their size, allowing for the effective separation and concentration of nanocrystals suspended in liquids. Additionally, the cross-flow ultrafiltration process can promote the alignment of nanocrystals through shear flow applied simultaneously to the transmembrane pressure [76], while filtration assisted by

ultrasound can modify the dynamics and orientations of nanocrystals during the transmembrane applied [77].

#### 3.2.2.1. Frontal filtration.
A method, called vacuum-assisted self-assembly (VASA), was used to fabricate CNC solid films [78]. Those films were fabricated using vacuum filtration of a 1.0 wt % CNC suspension through poly(tetrafluoroethene) filter paper on a porous ceramic support. Conducted at 0.09 MPa for up to 3 h, the vacuum filtration was followed by natural evaporation of residual water. The authors showed that the VASA method effectively aligned CNCs in an ordered manner by promoting their self-assembly into organized structures. The CNCs orientation in films was analyzed using wide-angle X-ray diffraction (WAXD), which tested CNCs alignment in various directions within the films. The resulting data revealed that films produced using this method exhibited a more uniform and structured CNCs orientation. Films with more aligned nanocrystals presented more vivid iridescence, precise color control, and uniform angular response. The VASA method thus enables precise control over CNCs orientation and enables to optimize the optical characteristics of iridescent films.

#### 3.2.2.2. Cross-flow filtration.
The process of cross-flow filtration, combined with transmembrane pressure, was used to fabricate CNCs membranes by deposition on support layers [79]. The authors exploited the change in the degree of alignment of the CNCs with the shear rate to deposit CNCs with different alignment on the surface of the support layer by varying the tangential flow velocity during ultrafiltration. They showed that a sufficiently high shear rate on the support surface was necessary for the CNCs to align in the direction of flow. To investigate the combined effect of suspension concentration on the shear-dependent flow alignment of CNCs, suspensions with different concentrations were pumped through a glass capillary tube with a diameter of 500 µm using a syringe pump at flow rates ranging from 1000 to 9000 µL·h⁻¹. As CNCs moved through the capillary, the shear stresses that developed on the capillary walls caused the CNCs to align, which was characterized by birefringence. The shear applied to the CNCs increased with increasing flow rates, resulting in a higher degree of alignment of the CNCs. Nanocrystal alignment on support layer was also characterized by evaluating the number of nanocrystals aligned in the flow direction for three different flow rates, i.e., 9, 30, and 80 mL/min. AFM phase images and angular histograms revealed that approximately 70–80% of nanocrystals in membranes formed at the highest flow rate (80 mL/min) were aligned parallel to the flow direction. In contrast, alignment decreased with lower flow rates, with about 50% alignment at 30 mL/min and random distribution at 9 mL/min. The order parameter, which measures alignment, was calculated based on the angle between the nanocrystals and the flow direction (see article for equation details). The order parameter values were 0.17, 0.48, and 0.7 for membranes prepared at flow rates of 9, 30, and 80 mL/min, respectively, indicating improved alignment with higher shear rates.

Recently, using a different cross-flow filtration setup equipped for SAXS measurements [80,81], we were able to assess the structural organization and orientation of CNCs near the surface of the filtration membrane. The SAXS patterns revealed an increase in the orientation level of the CNCs that accumulate near the membrane surface, resulting from the simultaneous effect of cross-flow and transmembrane pressure. For increasing filtration times, the SAXS patterns exhibited higher and higher scattering intensity in the vertical direction. This was due to the increase in concentration fluctuation in this direction. These fluctuations were induced by the regular orientation of CNCs, with their main axis orientated parallel to the membrane surface. By applying PCA to the SAXS patterns, the degree of alignment of CNCs could be monitored as a function of filtration time. SAXS experiments performed *in situ* during the filtration process revealed an increase in volume fraction with respect to the distance from the membrane surface over time. The increase in anisotropy observed in the SAXS patterns, especially at higher
filtration times and shorter distances from the membrane surface, linked
the concentration changes to the regular orientation of the CNCs in the
accumulated layers. By analyzing the concentration profiles alongside
the evolution of the anisotropy PCA profiles, we revealed that higher
CNC concentration near the membrane surface was associated with
increased alignment, and increased anisotropy correlated directly to
higher concentration in the concentration polarization layer.

**3.2.2.3. Filtration assisted by ultrasound.** With the aim of improving ultrafiltration performance, another set-up combining cross-flow ultra-filtration and ultrasound has been developed [77]. During cross-flow filtration assisted by ultrasound (US), the concentration of CNCs and starch nanocrystals close to the membrane was increased 10 times more than the feed suspension, with an increase in anisotropy 5 times that of the feed suspension. These results demonstrated that ultrafiltration methods (assisted by US or not) can be used to achieve a striking control over the colloidal concentration and orientation of nanocrystals.

In a recent study, conducted without filtration, we investigated the combined effect of US and shear flow on the orientation of CNCs in perpendicular directions, using time-resolved *in-situ* SAXS characterization [82]. To succeed, we developed a dedicated channel-type shear-flow/ultrasound cell to generate simultaneously a shear-flow-induced horizontal stress force and a US-induced vertical acoustic radiation force. For the first time, a vertical orientation of CNCs was observed, with their main axis (directors) aligned in the direction of ultrasonic wave direction of propagation. Concurrently, the application of shear flow induced a horizontal orientation of CNCs with their directors aligned along the velocity direction. When both forces were applied simultaneously, three regimes were observed. At low shear rates, US radiation predominated and oriented the CNCs in the direction of the acoustic wave propagation. At intermediate shear rates, an intermediate diagonal orientation of the CNCs was observed. Finally, at high shear rates, shear flow predominated, leading to CNCs orientation in the direction of flow. Building on these findings, the objective was to replicate the distinct orientation regimes observed in the first study simultaneously within a single sample, creating multiple layers with different CNC orientations. This motivated the development of a novel setup combining ultrasonic radiation and frontal ultrafiltration, compatible with *in-situ* SAXS observations. This enabled, for the first time, the observation of a multilayer orthotropic structuring of CNC suspensions, mimicking the organization of articular cartilage: a first layer composed of CNCs having their director aligned parallel to the horizontal membrane surface, a second intermediate isotropic layer, and a third layer of CNCs with their director vertically oriented along the direction of US wave propagation direction [83]. CNCs orientations obtained by this method are only transient in solution. Thus, further work is now needed to be able to maintain the oriented CNCs structures of interest.

In summary, filtration proves to be useful for aligning nanocrystals. The alignment of nanocrystals depends on several factors, including shear flow, transmembrane pressure, and the flow rate during the filtration process, as well as the simultaneous application of ultrasound. Although promising, these processes come with challenges. They can be complex to control and optimize, with precise adjustments needed for filtration parameters. More research is needed to make this method more cost-effective, scalable, and precise.

### 3.2.3. Calendering and solid-state drawing

Calendering is a process that consists in pressing a material between rolls in order to smooth and thin material sheets. When applied at high temperatures (typically ranging from 150 °C to 300 °C), this process is particularly effective for the manufacturing of viscoelastic thermoplastic material enabling the production of thin films. The versatility of calendering makes it suitable for various applications, including the production of flexible packaging materials and coatings [84]. Moreover,

unlike some more specialized techniques, calendering can be performed
at an industrial scale, making it a cost-effective solution for large-scale
manufacturing.

Using the calendering process in combination with solid-state stretching, Singh et al. evaluated the feasibility of orienting chitin nanocrystals within PLA nanocomposites for packaging applications [85]. The nanocomposite films were manufactured either by compression molding to form an isotropic material or by calendering to pre-orient the chitin nanocrystals within the film. Then, the films underwent solid-state drawing, which consists in stretching the films at a temperature below the melting point of the material to improve orientation, to orient the nanocomposite films. The authors compared the orientation, transparency, thermal properties, mechanical properties, and morphology of the two different films (i.e., compression molding and calendering) before and after applying solid-state drawing. The films were characterized by WAXS, which made it possible to define an orientation index for the chitin and PLA nanocrystals – the index varies from 0 (random distribution) to 1 (orientation in the same direction). This index is calculated by measuring the intensity of diffraction peaks at specific angles and comparing the maximum intensity with the baseline (see article for details). A higher value indicates better alignment of the nanocrystals. In the compression mold sample, the chitin nanocrystals were randomly oriented in the plane of the film. In this condition, the orientation index could not be calculated as no clear maximum intensity could be observed in the azimuthal integration of the chitin nanocrystals diffusion plane. This indicates that the chitin nanocrystals were randomly oriented in the plane of the film. On the contrary, the calendering process led to the partial orientation of the nanocrystals, with a reported orientation index of 0.8. The authors further demonstrated that the pre-orientation of the nanocrystals and the PLA by calendering favored their final orientation within the nanocomposites.

This approach for the production of materials with oriented nano-
crystals has been the focus of a single study to date, calling for more
investigation. In particular, there are several challenges to address for its
application in tissue engineering, including the lack of precise control
over particle concentrations during the process, as well as the high
operational temperatures that may be problematic for biological appli-
cations, potentially affecting the stability and viability of embedded
cells.

### 3.2.4. Unidirectional freezing

Unidirectional freezing is an advanced technique used to produce
low-density, porous structures with a highly ordered architecture [86].
This process consists in the controlled freezing of an aqueous suspension
or macromolecular solution along a specific, predetermined direction,
followed by the sublimation of the ice crystals. As the freezing front
advances, ice crystals nucleate and grow along the temperature
gradient. This crystal growth displaces the remaining liquid phase,
concentrating it in the yet-unfrozen regions. After freeze-drying, unidir-
ectonal porous networks, relevant for a wide range of applications in
tissue engineering (e.g., fluid transport, delivery), are obtained as a
direct consequence of the directional nature of the freezing process [87].

This method can be used to orient nanocrystals within a material. During the unidirectional freezing process, the nanocrystals can move toward the direction of freezing propagation. Indeed, ice crystals grow progressively in the same direction as the temperature gradient, creating a layered microstructure oriented in the direction parallel to the movement of the freezing front. During this process, nanocrystals are subjected to a shear flow field due to the difference in ice crystal growth rates in length and thickness, leading to their unidirectional orientation. This creates a material with a preferentially oriented structure, enhancing its properties in specific directions. For instance, Chu et al. used unidirectional freezing to fabricate an anisotropic hydrogel from CNCs [88]. They demonstrated that the isotropic CNC suspension can be aligned by shear flow in the vicinity of the ice crystals. Additionally, they showed that their hydrogels exhibited a hierarchical structure
perpendicular to the freezing direction.

While most studies on the unidirectional freezing of matrices con-
taining nanocrystals focused on pore orientation [89–91], this method
seems to enable the orientation of embedded nanocrystals as well,
opening new avenues of research. Achieving optimal alignment and
structure requires careful control of freezing parameters, nanocrystal
concentration, and polymer solution properties, making it a promising
yet complex method for fabricating materials with tailored mechanical
and functional properties.

3.3. Nanocrystal orientation using electric or magnetic fields

Organic nanocrystals can be oriented using an electric field through techniques such as electrospinning, and electro-compaction, each offering unique mechanisms to regulate the orientation and arrangement of nanocrystals within materials. Similarly, magnetic fields are also effective in orienting nanocrystals, allowing further control over their spatial organization.

3.3.1. *Electric field-based processes*

3.3.1.4. **Electric field.** Electric fields are widely used in materials engineering to align particles [92], fibers [93], and molecules [94] with dipole moments or polarizable structures, enabling directional properties in materials. Common applications include aligning liquid crystals in displays [95] and fibers in composites [96]. This technique is also applied to orient anisometric nanocrystals within composites.

Among the reported studies, one used electric field to control the orientation and structural organization of CNCs within cholesteric liquid crystalline phases in apolar solvents [97]. The electric field manipulation was shown to effectively align CNCs, enhancing their long-range order and providing microscopic homogeneity. By varying the electric field strength (0 to 600 V.cm⁻¹), the orientation of the CNCs could be precisely controlled, resulting in changes in the iridescent colors of the suspension, which are due to light diffraction. At lower field strengths, the CNCs aligned within the cholesteric phase, while higher fields induced a transition to a nematic phase, where the nanocrystals align along a common direction without forming the helical structure characteristic of cholesteric phase. The study also highlighted that electric field annealing (i.e., a process where a sequence of electric fields is applied over time), could be used to control CNCs alignment even after the field was removed, ensuring uniform orientation or inducing disorientation depending on the parameters of the electric field sequence. This work demonstrated that electric fields can be to dynamically control the arrangement of nanocrystals for applications in responsive materials, tunable optical devices, and nanocomposite design.

Another study explored the impact of electric field frequency on the orientation and pitch of CNC-derived lyotropic liquid crystals [98]. These CNCs were obtained from the interface between the isotropic and cholesteric phases of a CNC suspension. It was shown that, at low frequencies, the helix axis aligned parallel to the electric field, resulting in negative birefringence and structural changes such as helix pitch reduction (the reduction in the distance between successive loops of the helical structure). At higher frequencies, the helix axis aligns perpendicular to the electric field, leading to positive birefringence and pitch increase. This demonstrated that, while electric fields can induce reversible changes in the orientation and pitch of CNCs, these effects are more transient compared to those seen in the first study, where the alignment could be stabilized even after the field was removed. Thus, while electric fields can provide precise control over nanocrystal orientation, there are differences in the persistence of these effects depending on the experimental setup. In some cases, alignment can be maintained after field removal, whereas in others, the alignment remains reversible, depending on the strength, frequency, and duration of

the applied field.

Overall, positive aspects of the use of electric fields for nanocrystal alignment include precise alignment and control over structural properties. However, challenges include the need for specific conditions, such as the requirement for nanocrystals to be anisotropic and possess suitable surface properties (e.g., charge, polarity) for effective alignment. Moreover, the electric field strength and frequency must be carefully optimized to achieve desired effects, and maintaining long-term stability after field removal can be difficult. Despite these challenges, electric field manipulation holds great potential for creating responsive and adaptable materials.

3.3.1.5. Electrospinning. Electrospinning is a method used to produce fibers of different morphologies and diameters (ranging from tens of nanometers to several micrometers), while also enabling the formation of porous structures [99]. Electrospinning consists of an electro-hydrodynamic process in which a polymer solution is subjected to an electric field to produce a jet, followed by stretching and solvent evaporation to generate polymer fibers [99]. Electrospinning is commonly adjusted to enable the orientation of fibers for the design of anisotropic fibrillar scaffolds. For further insights, readers are referred to recent reviews discussing its historical development and futures prospects [100] as well as its applications in tissue engineering [101]. In the case of fibers containing nanocrystals, such as perovskite [102], chitin [103], or HAp nanocrystals [104], it was shown that the nanocrystals embedded in the fibers were mainly oriented along the fiber axis.

Among the nanocrystals studied for the design of nanocomposite fibers by electrospinning, CNCs are the most frequently used [103–106]. For example, one study reported the design of fibrous mats made of polyurethane (PU) and CNCs by electrospinning [105]. The effects of the CNCs concentration and collector rotation speed on the orientation of the fibers and CNCs were investigated. No alignment was observed for the slowest rotation (11 m.min⁻¹), while the fastest rotation (330 m. min⁻¹) resulted in fibers aligned in the direction of rotation. 2D-WAXS measurements provided access to molecular orientation and showed a preferential orientation of the embedded CNCs in the direction of the aligned fibers. In another study, similar CNCs orientation was observed, which was attributed to the repulsion between the surface-charged particles themselves, and to an alignment induced by stretching in the presence of an electric field during the electrospinning process [106].

To achieve optimal nanocrystal alignment in electrospun fibers, several key parameters must be controlled including the collector rotation speed, the polymer solution concentration, the applied voltage, and the distance between the needle and collector. Higher collector rotation speeds and higher polymer concentrations tend to favor better alignment by enhancing the stretching and orientation of the fibers during deposition. These factors facilitate the alignment of the nanocrystals along the fiber axis. However, several studies seem to contradict this hypothesis, suggesting instead a random orientation of CNCs in electrospun polymer fibers [107,108]. These contradictory results call for further work to confirm the spontaneous orientation of nanocrystals in electrospun fibers.

3.3.1.6. Electro-compaction. Electro-compaction is an electrochemical process used to align fibers, specifically developed to align collagen fibers, and has been applied in the fabrication of biomimetic scaffolds for tissue engineering, including tendons, corneas, nerves, and blood vessels [109]. This method consists in applying an electric current between two electrodes through a static fiber solution to align fibers. In the case of collagen fibers, this electric current generates a pH gradient in the solution such that the pH is approximately 3 at the anode and 11 at the cathode. The collagen molecules near the anode become positively charged and those near the cathode become negatively charged, with molecules gradually switching from positive to negative charges between the two poles. At the location corresponding to the isoelectric
point (i.e., electrically neutral state) of collagen, collagen molecules aggregate in an anisotropic structure in a highly organized manner. This process produces a scaffold in the form of a highly oriented wire or membrane.

Cudjoe et al. investigated the addition of CNCs to the electro-compaction process of collagen, expecting that charged nanocrystals would simultaneously align with collagen fibers [25]. To succeed, the authors functionalized CNCs with carboxylic acid groups or with both carboxylic acid and primary amine groups in order to produce a gradient of charged molecules between the anode and the cathode. Then, they applied voltage through a solution containing 95:5 wt% collagen/functionalized CNCs to form nanocomposite fibers via electro-compaction. To characterize the degree of CNCs alignment in the fibers, the authors used Raman spectroscopy. The intensity of Raman signals depends on the alignment of molecular bonds with the polarization of the laser. Thus, Raman can be used to characterize CNCs orientation, where only oriented CNCs are more strongly excited and have higher intensity signals at their specific Raman band (1095 cm⁻¹, corresponding to the distinct cellulose peak) [69]. Using this technique, the authors observed that electro-compacted composite fibers with 5 wt % CNC showed a 58% increase in intensity at 1095 cm⁻¹ compared to those aligned perpendicular to the laser. This result supports the successful co-orientation of CNCs in the electro-compacted collagen fibers.

Electro-compaction offers advantages such as the production of highly aligned fiber structures that enhance the mechanical properties and functionality of scaffolds for tissue engineering. It also enables the simultaneous alignment of nanocrystals with the fibers, potentially improving overall composite performance. However, it is important to note that the effectiveness of this method relies on the charge of the nanocrystals. Charged nanocrystals are more likely to be aligned under the electric field's influence, as their surface charge interacts with the field and helps orient them along with the fibers. On the other hand, non-charged or weakly charged nanocrystals might not align efficiently, as they are less responsive to the applied electric field. Moreover, optimizing the electric field strength, solution composition, and processing parameters, remains challenging. Additionally, the scalability of this method for industrial applications is not yet fully established, necessitating further research to explore other combinations of fibers and nanocrystals.

### 3.3.2. Magnetic field

A magnetic field arises from the motion of electric charges, such as those in an electric current, and creates a vector field that exerts forces on materials with magnetic properties. Materials react differently depending on their magnetic susceptibility: some are attracted (paramagnetic), others are repelled (diamagnetic), and some exhibit more complex properties (ferromagnetic). Magnetic field are commonly used to control the orientation of nanoparticles [110] and biological molecules like collagen [111], and so can be applied to nanocrystals.

Among the available types of nanocrystals, CNCs have a high aspect ratio and a negative diamagnetic susceptibility, allowing them to be manipulated using a magnetic field. The effect of weak magnetic fields (0–1.2 T) and concentration on the alignment of CNCs was studied [112]. The authors used CNCs at concentrations above and below the critical concentration (C*), which is defined as the concentration threshold beyond which CNCs transition from an isotropic state (random distribution) to a biphasic state where part of the CNCs are aligned. CNC alignment was characterized by SAXS, calculating the order parameter (S), which characterizes the CNC orientation distribution. An S of 0 corresponds to a random distribution (isotropic), an S of 1 indicates perfect alignment, and an S of -½ reflects perfect anti-alignment, where CNCs are perpendicular to the main direction. For CNC suspensions in water with a concentration below C*, no magnetic alignment was detected, regardless of the intensity of the magnetic field (S = -0.02). Conversely, for CNC suspensions with a concentration above C*, it was shown that a 0.56 T magnetic field allowed for nearly perfect alignment

of CNCs (S = -0.499), but it required 175 min to achieve. In comparison, a 1.2 T magnetic field induced alignment in a much shorter time. In these conditions, a partial alignment of CNCs was already observed in less than 2 min and a nearly perfect alignment (S = -0.499) was obtained after 129 min.

Another study confirmed these results and showed that a weak magnetic field (0.5 T) induced a preferential orientation of CNCs in the biphasic phase [113]. An additional study was carried out to compare the effect of a magnetic field (0.7 T) on CNC orientation in an organic (n-methylformamide) vs aqueous medium [114]. The authors also used the order parameter S calculated from SAXS measurement to characterize the alignment of CNCs. They observed a uniform partial order of CNCs (S of -0.20) in the organic medium under magnetic field, while a near perfect alignment of CNCs (S of -0.49) was obtained in water. The authors suggested that the higher order observed in the aqueous suspension could be attributed to its lower viscosity and fluid-like behavior, in contrast to the organic medium suspension, which exhibited elastic gel-like properties.

CNC-based thin films find applications in coatings and cosmetics [115,116]. For these applications, the photonic properties of CNCs are important, and controlling CNCs orientation is necessary to design thin films with appropriate angular optical responses. In this context, magnetic fields were used to control the orientation of the cholesteric domains of CNCs within CNC-based thin films [117]. Here, the authors placed a CNC suspension in a petri dish and left it to dry at room temperature. Then, they placed the drying suspension in immediate vicinity of small commercial magnets (≈ 0.5 - 1.2 T). To capture the angular dependence of the optical response, the films were analyzed using angular resolved optical spectroscopy, which involves illuminating the sample with a collimated light beam at a specific angle. The films prepared without magnetic field exhibited a broad angular wavelength response, suggesting a wide distribution of cholesteric structure orientations within the film. In contrast, the films prepared with a vertical magnetic field showed strong specular reflection in the direction perpendicular to the sample surface, indicative of a cholesteric structure that is well aligned throughout the film thickness. Together, the authors demonstrated that it is possible to orient the cholesteric structure of CNCs using a magnetic field, resulting in an oriented material.

The alignment of particles using magnetic fields offers several advantages, including precise, uniform, and rapid control over their orientation. This technique is applicable to a wide range of materials, from colloidal suspensions [118] to biomacromolecules [119]. Among these, nanocrystals have gained particular interest due to their distinct mechanical, optical, and structural properties that can be tailored by orientation. While current studies predominantly focused on CNCs, extending this technique to other types of nanocrystals could enhance their potential. In particular, materials such as Hap [120], chitin [121] and cerium oxide [122] have not yet been studied in their nanocrystalline form with magnetic fields.

## 4. Materials with oriented nanocrystals for tissue engineering

Many biological systems feature hierarchically ordered, composite structures with elements spatially oriented up to the macroscopic scale, such as cornea [123], articular cartilage [9], skeletal muscles [11], blood vessels [124], skin [125], bone [126], ligaments [127], intervertebral disc [12], and tendons [10]. Controlling nanocrystal orientation at the nanoscale can influence polymer network formation and propagate alignment across length scales, ultimately imparting anisotropic mechanical properties and structural features that better mimic native tissues. The anisotropic organization of tissues plays an important role in their biomechanics and biological functions. Among other functions, the anisotropic organization of tissues contributes to guiding cell differentiation, growth, migration, and tissue regeneration [128–130]. The hierarchical structure of tissues is affected by injury and diseases, and regenerating or reproducing such structures is a challenge
[131]. In tissue engineering, the use of materials containing oriented nanocrystals enables to mimic the hierarchical structures of biological tissues. To date, these materials have been mainly investigated for two applications: mechanically reinforcing scaffolds, and guiding cell alignment. Among the different materials studied, hydrogels are of particular interest due to their high water content and cytocompati-bility, making them ideal for mimicking the extracellular matrix.

## 4.1. Mechanical reinforcement of material scaffolds

Hydrogels are hydrated 3D molecular networks. They are often used for tissue engineering applications because they can best recapitulate the physical and biochemical properties of living tissues [132–134]. However, despite their cytocompatibility and tunability, their mechanical properties are often insufficient to mimic or replace load-bearing tissues, such as cartilage or bone. For instance, cartilage has a Young's modulus around 1 MPa [135], while that of bone is in the range of 10 GPa [136], which hydrogels generally do not reach. To overcome this challenge, mechanical reinforcement strategies are necessary. One promising approach involves incorporating nanocrystals, and more specifically, orienting these nanocrystals within the hydrogel matrix to enhance their mechanical properties.

Using non-directional methods, it has been demonstrated on several occasions that the presence of nanocrystals increases the mechanical properties (tensile modulus, compressive modulus, strength, toughness) of materials. For example, in a polylactic acid (PLA) scaffold obtained by polymerization, the addition of a small amount (0.8 wt %) of CNCs increased the compressive modulus of the material by 368% compared with the scaffold without nanocrystals [137]. Other studies reported that the mechanical properties of scaffolds increased with increasing concentration of CNCs using cross-linking [138], casting [139,140], or even freeze-drying [141]. The addition of starch nanocrystals also increases the mechanical properties of materials. Typically, Velásquez-Castillo et al. prepared cassava starch (CS) films containing quinoa starch nanocrystals (5% w/w) by casting and showed that the tensile strength and Young's modulus of the films increased by about 2.5 times in the presence of quinoa starch nanocrystals [142]. The addition of chitin nanocrystals to a chitosan-based porous scaffold also increased the mechanical properties of the scaffold [33]. However, nanocrystal-embedded materials often fail to achieve the mechanical properties desired for a chosen application. This could be addressed by aligning nanocrystals using various techniques.

### 4.1.1. Mechanical reinforcement using extrusion-based 3D printing

The integration of various reinforcing materials during the extrusion process significantly enhances the performance characteristics of materials [143]. One promising approach is the use of nanocrystals, which can be aligned during the printing process to further improve mechanical strength and functionality.

To create materials for the production of objects mechanically reinforced with aligned CNCs via extrusion-based 3D printing, visco-elastic inks composed of CNCs in a photopolymerizable monomer solution were developed, enabling the production of cellular architectures with customized mechanical properties for biomedical applications [144]. First, to develop high CNC-loaded composite architectures, monomer-based inks composed of 10–20 wt% CNCs, 2-hydroxyethyl methacrylate, polyether urethane acrylate, and a photoinitiator, were investigated for UV light curing after printing. Then, CNC alignment during printing was confirmed by optical microscopy and 2D-WAXS measurements, showing improved CNC orientation in printing samples compared to solution-cast films. Two types of polyether urethane acrylate were tested to produce soft and stiff composites. The authors studied the CNC alignment in 3D printed polymer composites and its effect on mechanical properties. They showed that CNC alignment along the printing direction significantly enhanced mechanical properties, particularly in the longitudinal direction (Young's Modulus $E \approx 1.7$ GPa

in the direction of CNC alignment, vs $E \approx 1.4$ GPa in the direction perpendicular to CNC alignment), due to improved load transfer. This effect was more pronounced at higher CNC content, and even more for stiff composites.

Using this approach, the same authors later developed a method to fabricate complex 3D architectures with increased stiffness and programmable shapes, i.e., objects that can bend or twist upon hydration [145]. To control and modulate the mechanical strength and swelling direction of the printed hydrogels with humidity, CNCs were added to a photocrosslinkable viscous solution containing N-isopropylacrylamide (NIPAM), an ethylene glycol dimethacrylate (EGDMA) crosslinker, and a photoinitiator (Irgacure 2959). The solution was 3D printed, which induced CNC alignment, before UV photocuring the composite materials (Fig. 3). To investigate the effect of CNC alignment on mechanical properties, they measured the stiffness of hydrogels with CNCs aligned in the longitudinal or transverse direction relative to the applied load. The Young's modulus increased from approximately 70 Pa for the NIPAM hydrogel alone to 16.6 kPa for the NIPAM hydrogel containing aligned CNCs (20 wt%), constituting a 236-fold increase in stiffness. Furthermore, the average ultimate stress properties of the composites, i. e., the maximum stress the material can withstand before breaking, was approximately twice higher in the longitudinal direction (parallel to the CNC alignment) than that in the transverse direction (perpendicular to the CNC alignment). Interestingly, the authors further demonstrated that hydrogel swelling could be precisely controlled in space by adjusting CNC concentration and alignment. In this application, locally printing more concentrated and more aligned CNCs led to more pronounced anisotropic swelling. These adjustments enabled the fabrication of objects capable of 4D morphing, i.e., changing shape over time in response to external stimuli, such as humidity or temperature.

In the context of bone tissue engineering, another study reported the use of extrusion-based 3D printing for the production of PLA scaffolds mechanically reinforced by oriented CNCs [146]. Tensile mechanical tests were performed on the resulting composites. An increase in the Young's modulus of the PLA fibers was observed with the addition of CNCs (3 wt%), from $2.4 \pm 0.1$ GPa to $3.1 \pm 0.2$ GPa. Interestingly, when the authors increased the CNC concentration from 3 to 5 wt%, they observed a decrease in the Young's modulus of the nanocomposite fibers, even falling below that of pure PLA fibers. Supported by the literature [147], the authors hypothesized that CNCs were properly distributed and aligned only at low concentrations. At low CNC concentrations, nanocrystals would successfully align with PLA chains upon printing, which would allow for the applied stress to be transmitted from one fibril to the next within the composite, thus leading to a uniform distribution of stress throughout the material. Conversely, at higher CNC concentrations, CNCs would disperse poorly in the PLA matrix and would not be properly oriented in the direction of the PLA chains when printed, which would hinder mechanical reinforcement.

Overall, extrusion-based 3D printing offers flexibility in designing complex structures with various nanocrystals types. While it enables the production of customized scaffolds, this process may result in some inconsistencies in mechanical reinforcement due to the challenges associated with achieving uniform alignment and distribution of the nanocrystals throughout the material.

### 4.1.2. Mechanical reinforcement via other methods

Apart from 3D printing, other orientation methods have demonstrated the benefits of orienting nanocrystals to enhance mechanical properties. In our laboratory, we investigated the possibility of reinforcing mechanically a polyethylene glycol diacrylate (PEGDA) hydrogel with oriented CNCs using an ultrafiltration system combined with UV curing [148]. We filtered a CNC (2 wt%)/PEGDA (70 wt%) suspension for 0, 24, and 66 h before UV curing to obtain 1-mm thick nanocomposites. We characterized CNCs orientation by birefringence, SEM, and SAXS, showing an increase in CNC orientation with increasing filtration time, and no orientation without ultrafiltration. The tensile
mechanical properties of these films were then evaluated, revealing an increase in Young's modulus from 6.76 ± 1.19 MPa (0 h) to 58.67 ± 11.81 MPa (24 h), and up to 112 ± 4.60 MPa (66 h). Two reasons have been put forward to explain the superior mechanical properties of filtered nanocomposites: an increase in CNC concentration, which leads to increased interactions with the PEGDA network and thus an increase in material rigidity; and the orientation induced by nanocrystal filtration. To verify this, tensile tests would have to be carried out in the direction perpendicular to the CNC orientation. Ultrafiltration provides precise control over nanocrystals alignment. Moreover, it allows higher concentrations to be achieved compared to other techniques, enhancing the mechanical performance of nanocomposites in tissue engineering applications.

Similar mechanical reinforcement was obtained for composite fibers,

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Impact of CNC alignment via 3D printing on the compressive properties of CNC/NIPAM hydrogels. (A) Synthesis and 3D printing process, including ink formulation, extrusion, and nanocrystal alignment. (B) CNC orientation in 3D-printed NIPAM hydrogels, visualized by 2D-WAXS azimuthal intensity distributions for different nozzles diameters (840 and 410 µm). (C) Mechanical properties comparison of neat NIPAM hydrogels and CNC–NIPAM hydrogels (20% and 25% CNCs), under compression, including (a) stress-strain curves, (b) Young's modulus, and (c) ultimate stress in longitudinal and transverse directions at 70% strain, with error bars indicating standard deviation (n = 6). Adapted from Fourmann et al. [145].</figcaption></figure>

using magnetic induction. For example, a strategy consisted in adsorbing Fe₃O₄ nanoparticles onto the surface of CNCs before dispersing them in a PLA solution, to allow for their magnetic alignment within the PLA matrix under magnetic field (≈ 50 - 60 mT) [149]. The films were then obtained by drying the PLA solution containing the CNCs. Compared to non-oriented CNCs, it was shown that orienting CNCs (1 wt%) parallel to the magnetic field increased the tensile strength and percent elongation of the composite by about 70% and 240%, respectively. In contrast, the perpendicular orientation of CNCs led to increases in tensile strength and percent elongation of only approximately 58% and 172%, respectively. The parallel orientation yielded greater improvements in mechanical properties compared to the perpendicular alignment. This is likely because the parallel orientation of CNCs with the tensile direction of the PLA matrix allows the film to handle higher stress and strain while promoting greater deformation before failure. In comparison, while magnetic induction effectively aligns CNCs and enhances mechanical properties, it is limited to nanocrystals with magnetic properties.

A study also reported the use of calendering for the enhancement of the mechanical properties of PLA fibers via the embedding of oriented chitin nanocrystals [85]. In the alignment method described, PLA-chitin nanocrystals nanocomposite films were flattened by calendering to pre-orient the nanocrystals on the PLA fiber surface, followed by tensile drawing to stretch and further align the nanocrystals in a specific direction. The authors demonstrated that the pre-orientation of chitin nanocrystals significantly increased the mechanical properties of their PLA nanocomposites, with tensile modulus, strength, elongation at break, and toughness increased by 74%, 360%, 2400%, and 9500%, respectively. Calendering has the advantage of orienting nanocrystals more efficiently and producing smooth, uniform surfaces. However, it is limited to flat shapes and may require higher temperatures, which could degrade heat-sensitive materials.

Finally, electro-compaction was successfully used to mechanically reinforce collagen fibers with CNCs (5 wt%) (Fig. 4) [25]. As aforementioned, with this system, concentration can affect CNCs alignment, with a decrease in alignment above a CNC concentration between 5 and 10 wt%. In line with these results, the authors found that mechanical reinforcement, as indicated by the Young's modulus, increased with CNC content up to 5 wt% (230 MPa), but then decreased for higher concentration (135 and 91.5 MPa for 7.5 and 10 wt% CNC, respectively). Electro-compaction provides efficient nanocrystal alignment and uniform reinforcement, enhancing the mechanical integrity of simpler shapes. However, it is less versatile in creating complex geometries compared to other techniques, making it particularly suitable for applications where the focus is on reinforcing less intricate structures in tissue engineering.

Overall, the orientation of nanocrystals has been shown to be a key factor in improving the mechanical properties of materials, with all methods explored successfully achieving mechanical reinforcement. However, few studies have evaluated these materials *in vitro* or *in vivo*, especially not in a context where mechanical reinforcement is necessary. Addressing this gap is crucial, as it will provide a more comprehensive understanding on how these reinforced materials perform in real biological contexts and their potential for tissue engineering applications.

## 4.2. Cell alignment

Cell alignment is an important feature of many living tissues including muscle, vascular, and nerve tissues [150,151]. In these tissues, cell alignment, in combination with cell proliferation, migration, and secretion, determines tissue organization and defines its physical properties and biological functions. It has also been shown that cell alignment influences cell differentiation, such as the differentiation of human tendon stem cells [152], and the alignment of the collagen fibers they produce [153]. Cell alignment is also essential for tissue repair and regeneration as it helps minimize scar formation and restore normal function to damaged tissues [154,155]. Therefore, it is believed that
biomaterials, particularly hydrogels, that can promote cell alignment could help improve tissue repair. Several methods exist to orient and align cells in hydrogels [156], including techniques based on electro-magnetic fields [157], chemical gradients [158], and micro-scale oriented patterns [159]. Additionally, methods such as sintering, which primarily serves to densify powders, can also be used to micro- or nano-texture surfaces, influencing cell alignment indirectly [160]. Surface etching [161], and femtosecond laser [162] texturing have also been shown to effectively align cells. However, their applicability to hydrogels remains limited due to challenges related to the material's structure and composition. In this context, hydrogels containing oriented nanocrystals are gaining increasing attention.

### 4.2.1. Cell alignment through the magnetic orientation of nanocrystals

In the context of muscle tissue engineering, De France et al. sought to simulate native muscle tissue environments with an injectable anisotropic hydrogel that could guide the alignment of differentiated skeletal muscle myotubes [163]. Using magnetic orientation, they first designed a poly(oligoethylene glycol methacrylate) (POEGMA) hydrogel containing aligned CNCs. In this system, a mixture of CNCs, hydrazide- and aldehyde-functionalized POEGMAs was exposed to a magnetic field (0–1.2 T) during gelation to orient CNCs. SAXS analysis revealed no preferential CNC orientation without a magnetic field, but a 1.2 T magnetic field induced a lasting alignment of CNCs. The authors investigated the effect of CNC magnetic alignment on the terminal differentiation of C2C12 myoblasts into highly oriented myotubes. They used hydrogels that exhibited the highest degree of alignment (corresponding to the hydrogels with the highest CNC concentrations). First, they started with 2D experiments where myoblasts were placed on hydrogel constructs gelled in the presence or absence of a 0.56 T magnetic field. After 2 days of culture, no apparent differences were observed in the morphology or orientation of the myoblasts between the magnetically aligned hydrogels and the control hydrogels. However, upon transitioning to a differentiation medium and extending the culture period to 8 days, it was observed that the myotubes developed on magnetically aligned hydrogels exhibited a highly oriented morphology. The observed cell alignment was attributed to the alignment of CNCs

within the hydrogel and, thus, demonstrated the effect of the magnetic alignment on the later stages of cell differentiation. Following these 2D experiments, 3D experiments were conducted on C2C12 myoblast cells encapsulated in the hydrogels. The viscoelastic hydrogels were then extruded in the presence or absence of a magnetic field and incubated using the same differentiation protocol as for the 2D experiments. The authors observed cell orientation only in aligned hydrogels. Moreover, the encapsulated cells remained viable throughout the experiment, which validated the cytocompatibility of the approach. Interestingly, cells accelerated the hydrogel degradation, which was attributed to their enzymatic activity. This process highlights the cells' ability to not only utilize the hydrogel matrix initially for alignment but also to remodel their microenvironment over time. Overall, this study presented an innovative method for creating anisotropic hydrogels that can direct the growth and alignment of myotubes. Interestingly, this system is injectable, possibly paving the way for a new generation of muscle regeneration treatments. However, the study was limited to a short-term evaluation (2 to 8 days of culture); and long-term data would be necessary to demonstrate the functionality of the aligned myotubes.

Another study described a similar approach to guide the alignment of human adipose-derived stromal cells (hASCs) and mimic the cell organization of anisotropic tissues such as tendons [164]. The authors combined an enzymatically cross-linked gelatin hydrogel with CNCs (0.1, 0.2, and 0.3 wt%) that were decorated with magnetic particles to produce hydrogels containing magnetically aligned CNCs. Then, they performed both 2D (seeding) and 3D (encapsulation) cell culture experiments. For the 2D experiments, the authors demonstrated that after 3 days of culture, cells seeded on anisotropic hydrogels with CNCs concentrations of 0.1 and 0.2 wt% showed directional growth aligned with the nanocrystals, whereas cells on isotropic hydrogels did not show such alignment and displayed more random distribution. For the 3D experiments, using anisotropic hydrogels with CNCs concentrations of 0.1 and 0.2 wt% also led to preferential alignment, which was not observed in isotropic hydrogels. Notably, a CNCs concentration of 0.3 wt% did not lead to cell alignment. The authors concluded that the specific dimensions and/or spacing between magnetically aligned nanoparticles are crucial for achieving cell alignment. Using a similar methodology,

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Fabrication and mechanical properties of collagen/CNC nanocomposite fibers. (A) Schematic of the electro-compaction alignment process of collagen (red) and t-CNCs (green) fibers. (B) SEM images of dry cross-linked fibers with 0, 2.5, 5, 7.5, and 10 wt% t-CNC—COOH—NH₂. (C) Mechanical characterization of wet cross-linked fibers: (a) stress-strain curves and (b) Young's modulus (N = 10/group). Adapted from Cudjoe et al. [25].</figcaption></figure>
the same authors later developed a bilayered anisotropic hydrogel to mimic the tendon-bone tissue interface (Fig. 5) [165]. The authors produced the first layer by forming CNC-containing, gelatin hydrogels in molds under the influence of a range of uniform magnetic fields (200 to 400 mT). Then, a mineralized isotropic phase composed of HAp nanocrystals and gelatin was casted on top of the first layer to produce the desired bilayer system. Using confocal microscopy to characterize the cytoskeletal organization of encapsulated hASCs, the authors observed a preferred cell orientation in anisotropic hydrogels after 3 days, which was maintained after 1 week. Although the study did not directly investigate cell adhesion, the authors hypothesized that the nanotopographical cues from the aligned nanoparticles may enhance adhesion and promote cell spreading in one direction. Since no magnetic field was applied during the entire culture period, the authors concluded that the observed cell alignment after 7 days was due to the intrinsic anisotropy of the hydrogels, specifically the alignment of CNCs. This article offers a novel approach to integrating mineralized and anisotropic features in hydrogels for enhancing the interaction between different types of tissues. However, questions remain regarding how well the alignment of CNCs and the use of hASCs in gelatin can truly mimic the complex, structural and functional characteristics of tendons.

### 4.2.2. Cell alignment through nanocrystals alignment by filtration

A study reported the development of therapeutic contact lenses that are capable of releasing a nonsteroidal anti-inflammatory drug (diclofenac sodium DFN) [21]. To succeed, the authors used the vacuum filtration method (see section 2.1.2) to develop a corneal stroma-mimicking film composed of collagen and dialdehyde CNCs, with CNC alignment replicating the natural orientation of keratocytes in corneal stroma. In the proposed system, CNC alignment was used not only to mimic the transparency and mechanical functions of the aligned keratocytes but also to investigate how such anisotropy could guide cell alignment and growth on the material's surface, for potential in vivo applications (Fig. 6). The films were prepared by filtering a solution containing collagen and dialdehyde-modified CNCs through a porous ceramic support associated with a cellulose ester filter membrane (0.22 µm pore size). The authors observed that the dialdehyde-modified CNCs formed a layered structure upon the slow filtration process, with layers parallel to each other and oriented perpendicular to the top surface. Collagen also organized into fibrils oriented parallel to the film surface, as characterized by SEM. A morphological analysis was performed 72 h after seeding NIH 3T3 cells (i.e., a fibroblast cell line) either on glass slides (control) or on films containing oriented nanocrystals. The alignment of 3T3 cells on different films was quantified using SEM aging to measure cell morphology (length and width) and orientation. The orientation of cells was further quantified by plotting the angle distribution of cell growth. Statistical analysis of the SEM images showed that cells on collagen/dialdehyde CNCs films had increased elongation and were more aligned along the direction of the fibrils, with a narrower angle range of cell orientation compared to the more random distribution observed on control films. This result suggests that the oriented films led to a more aligned and extended fibroblast morphology in vitro, but the study lacks an in vivo analysis to confirm whether this effect is valid in a more complex biological environment. Furthermore, while the study focused on fibroblast alignment, it is unclear whether this behavior can be extended to more relevant corneal cells, such as keratocytes, which are crucial for reproducing the full biological functionality of corneal tissue.

Overall, these studies showed that anisotropic hydrogels can promote cell alignment, both in 2D (cell seeding) and 3D (cell encapsulation) experiments. Cell alignment was systematically linked to the preferential orientation of nanocrystals within the hydrogels, highlighting the potential of these materials for specific biomedical applications.

### 4.3. Perspectives

Tissue engineering is a constantly evolving field, where new technologies and methods are continuously explored to improve the quality and functionality of fabricated tissues [166–169]. Among these technologies, 3D printing, ultrafiltration, electrospinning, electro-compaction, unidirectional freezing, and magnetic induction allow for the orientation of nanocrystals within matrices. Although few studies have been conducted on this topic, they all agree that the orientation of nanocrystals within biomaterials directly influences the mechanical properties of the fabricated composites, as well as the orientation of cells in contact with these materials. This is a new area of research and much remains to be done.

To date, materials containing oriented organic nanocrystals for tissue engineering have been mainly based on the use of CNCs due to their low cost and availability, favorable mechanical properties, and cytocompatibility. However, it would be beneficial to expand this research to other types of nanocrystals, such as chitin, starch, and even to inorganic nanocrystals such as hydroxyapatite (HAp) and cerium oxide nanocrystals. Each type of nanocrystal offers unique properties that could enrich the field of tissue engineering. For instance, chitin nanocrystals can be used as antibacterial agents for wound healing applications [170], and adding orientation to these nanocrystals could further enhance the mechanical properties of the materials. On the other hand, starch nanocrystals are biodegradable and can be used to gradually release drugs or growth factors, thereby promoting tissue regeneration [171]. Cerium oxide nanocrystals possess remarkable antioxidant properties, which can be beneficial for reducing oxidative stress in damaged tissues and promoting healing, thus enhancing cell viability and functions [172]. HAp nanocrystals are particularly promising for bone regeneration due to their similarity to the mineral components of human bones [173]. Therefore, controlling the orientation of HAp nanocrystals embedded in materials would allow for mimicking the anisotropic structure of natural bone tissues, thereby improving the functionality of implants. Given concerns about the cytotoxicity of HAp nanocrystals at certain concentrations, further research is also necessary to optimize their safe use in clinical applications. By broadening research to include these various nanocrystals, we could develop more specialized and cytocompatible materials for diverse clinical applications. In addition to the cytocompatibility of the nanocrystals themselves, the polymer matrix in which the nanocrystals are embedded also plays a critical role. Therefore, the selection of a suitable polymer matrix is essential to ensure that the nanocrystal-containing composites is cytocompatible and promote cell adhesion, proliferation, and differentiation while maintaining structural integrity over time.

As highlighted in this review, only a handful of biomaterials containing oriented nanocrystals have been used in vitro, and, to our knowledge, none have been used in vivo. Interestingly, despite the variety of manufacturing methods available, the few examples of concrete biomedical applications that we found are all based on filtration or magnetic induction. Increased research into other technologies such as 3D printing, electro-compaction, and ultrafiltration, could provide innovative solutions and further improve the mechanical properties of materials designed to mimic natural tissues, while promoting cell alignment and other functions. For example, 3D printing allows for precise customization and fabrication, including the possibility of cell encapsulation [174], but its potential for orienting nanocrystals in scaffolds remains underexplored. Similarly, electro-compaction and ultrafiltration could be used to produce gradients of mechanical properties and specific structural alignments, thereby optimizing conditions for cell proliferation and migration [175,176]. In summary, further research in these areas could not only improve the fabrication of biological tissues but also open new perspectives for more effective clinical applications and personalized treatments.

In combination with nanocrystal alignment, it will be interesting to apply the described manufacturing methods to align other elements such
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Effect of hydrogels containing magnetically aligned CNCs on mechanical reinforcement and cell alignment. (A) Magnetic field alignment of CNCs. (B) Polarized optical microscopy images during cross-linking with or without a 200 mT magnetic field (scale bars = 1 mm). (C) Young's Modulus from strain-stress curves under compression forces shows *p* < 0.05 for isotropic hydrogel with the same CNC concentration, and #p < 0.05, ##p < 0.01 for hydrogels under the same magnetic field. (D) Biological characterization of 3D nanocomposite hydrogels using encapsulated hASCs after 21 days of culture: confocal images of TNC (green), nuclei (blue), and cytoskeleton (red) (scale bars = 200 µm). Adapted from Echave et al. [165].</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Impact of vacuum-filtrated CNC/collagen films on cell morphology and alignment. (A) SEM images of pure CNC films (a), dialdehyde CNC films (b), pure collagen (c), and composite films (d-g). (B) SEM images of 3T3 cells cultured on glass slide (a), and composite films (b-e) (scale bar = 5 µm). (C) Quantification of 3T3 cell length, width (a), and orientation angle distributions on the glass slide (b) and composites films (c). Adapted from Xu et al. [21].</figcaption></figure>
as fibers, porous structures and cells, thus expanding the possibilities for
design and functionality. Fiber orientation, which can be obtained by
electrospinning [177], soft lithography [178], or magnetic induction
[111], can strengthen tissue resistance and structure [179]. This is
crucial for applications such as muscle regeneration or the reconstruc-
tion of connective tissues, where mechanical strength is essential [180,
181]. In addition, the orientation of fibers can help guide and direct cell
growth [182]. Similarly, pore alignment, which can be obtained mostly
by directional freezing [183], can promote efficient diffusion of nutri-
ents, growth factors, and cell waste throughout fabricated tissues
[184–186]. This may be necessary to ensure the long-term viability and
functionality of encapsulated cells. This can also be beneficial in the
fabrication of vascular tissues as it can facilitate vascularization [186],
or nerve tissues as it could favor axon regeneration [187].

Beyond the development of new materials with an oriented struc-
ture, it seems crucial to rapidly demonstrate the potential of these ma-
terials for biomedical applications through concrete *in vitro* and *in vivo*
experiments. Additionally, the precise alignment of nanocrystals could
play a crucial role in the orientation and migration of cells, thereby
facilitating tissue regeneration and integration within the human body
[154,155]. While some studies have focused on these aspects, future
research could also investigate how nanocrystal orientation influences
the adhesion and differentiation of cells, particularly stem cells. Given
the impact of mechanical properties on cell responses [188,189], it is
likely that the alignment of nanocrystals affects these processes,
potentially opening new avenues for enhancing tissue regeneration.

5. Conclusion

This review article highlights the growing interest in orienting
nanocrystals within composite materials for tissue engineering appli-
cations. Although many systems have been developed, only a few have
achieved concrete biomedical application, and only relatively pre-
liminary biological studies have been carried out. More work is expected
to expand the variety of nanocrystals used, in combination with new
orientation methods. More importantly, significant translation work
remains to be done. This includes further investigation of the effect of
anisotropic materials on cell adhesion, differentiation, and migration,
and advancing the use of these materials *in vivo*. Only a greater dialogue
between materials engineers, biologists and clinicians will pave the way
for the proper design and application of this new generation of aniso-
tropic biomaterials.

CRediT authorship contribution statement

**Fanny Bosson:** Writing – review & editing, Writing – original draft, Conceptualization.
**Nicolas Hengl:** Writing – review & editing, Supervision.
**Baptiste Charbonnier:** Writing – review & editing.
**Jérôme Guicheux:** Writing – review & editing.
**Frédéric Pignon:** Writing – review & editing, Supervision.
**Vianney Delplace:** Writing – review & editing, Writing – original draft, Supervision, 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

The authors are grateful to the I-MEP² Doctoral School (Engineering - Materials, Mechanics, Environment, Energy, Processes, Production) from the University Grenoble Alpes for the financial support of FB's PhD. The French Agence Nationale de la Recherche (ANR), under grant ANR-20-CE43-0015 (ANISOFILM) is also acknowledged for financial support. 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).

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