Elsa Mazari-Arrighi

Dmitry Ayollo

Wissam Farhat

Auriane Marret

Emilie Gontran

Pascale Dupuis-Williams

Jerome Larghero

Francois Chatelain

Alexandra Fuchs
email: alexandra.fuchs@cea.fr

Construction of functional biliary epithelial branched networks with predefined geometry using digital light stereolithography

Keywords: Tissue engineering, bile ducts, biliary tree, 3D bioprinting, DLP stereolithography, cholangiocytes

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Liver tissue engineering is an actively developing research area in the field of drug screening to develop more physiologically relevant toxicity assays and models of liver diseases. Furthermore, it is a promising solution for regenerative medicine to alleviate the need for donor liver tissue. In recent years, significant progress has been made in culturing primary or iPSC-derived hepatocytes in the form of spheroids or organoids to achieve higher differentiation and maintain better functionality in vitro [START_REF] Bell | Comparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Longterm Toxicity Applications: A Multicenter Study[END_REF]- [START_REF] Hu | Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids[END_REF]. To date, the most advanced example of this approach was described by Takebe et al, who managed to produce liver buds composed of iPS-derived endoderm, endothelial and mesenchymal cells grown to form organoids that were shown to rescue hepatic functions in a mouse model of acute liver failure [START_REF] Takebe | Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells[END_REF]. Such an approach has the potential to create relevant models of metabolic liver diseases, as well as to ultimately treat the partial failure of liver functions. However, they do not take into account the critical role played by the bile ducts and ductule network in collecting and clearing toxic compounds and metabolites from the hepatic tissue.

The human biliary tree consists of the intrahepatic and extrahepatic sections. The intrahepatic biliary tree is composed of ductules that collect and modify the bile from the bile canaliculi formed by hepatocytes in the liver lobules. The intrahepatic ductules gradually merge into the common hepatic duct which leads to the extrahepatic biliary tree [START_REF] Tabibian | Randomised clinical trial: vancomycin or metronidazole in patients with primary sclerosing cholangitisa pilot study[END_REF], [START_REF] Nakanuma | Pathology and pathogenesis of intrahepatic bile duct loss[END_REF]. Over the past few years, there have been a few successful attempts to produce bio-artificial bile ducts (BABD). For example, in 2019, Tysoe et al. have described a way to build a bile duct with a lumen diameter of 250 µm using a rolled polyglycolic acid or a collagen hydrogel layer as a scaffold for cholangiocytes isolated from murine liver and amplified as organoids [START_REF] Tysoe | Isolation and propagation of primary human cholangiocyte organoids for the generation of bioengineered biliary tissue[END_REF]. Another approach was described by Chen et al. in 2018 in which authors used cholangiocyte-like cells from the murine liver to seed the outer surface of polyethersulfone hollow fiber membranes functionalized with L-Dopa and collagen [START_REF] Chen | Bioengineered bile ducts recapitulate key cholangiocyte functions[END_REF]. The result was a BABD with an inside-out inverted polarity and a diameter of 1 mm. While the above-mentioned works mark obvious progress in extrahepatic bile duct reconstruction, reproduction of the intrahepatic part of the biliary tree is more challenging due to both its size and its complexity. Indeed, the intrahepatic biliary tree permeates every level of liver anatomy from the hepatic hilum to the liver lobule, with internal luminal diameters ranging from 800 µm in the interlobular ducts down to 10 µm inside the lobules.

Using the relatively popular top-down approach of decellularization and subsequent recellularization of the whole organ, the liver vasculature has been successfully repopulated with endothelial cells [START_REF] Baptista | The use of whole organ decellularization for the generation of a vascularized liver organoid[END_REF]. However, the recellularization approach for the biliary tree showed limited success [START_REF] Hassanein | Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold[END_REF]. This may be due to several factors ranging from the intricate biology of cholangiocytes to the tortuous and complex architecture of the intrahepatic biliary tree.

Bottom-up approaches to de novo engineering of the intrahepatic biliary tree have been pursued using hydrogel matrices. In vitro, extracellular matrix (ECM) substitutes such as type 1 collagen or Matrigel (an ECM hydrogel produced by the mouse Engelbreth-Holm-Swarm sarcoma cell line) have shown promising results both for cholangiocytes alone or in co-culture with hepatocytes [START_REF] Katsuda | Biliary epithelial cells play an essential role in the reconstruction of hepatic tissue with a functional bile ductular network[END_REF], [START_REF] Auth | Morphogenesis of primary human biliary epithelial cells: induction in highdensity culture or by coculture with autologous human hepatocytes[END_REF]. Cholangiocytes, known to have a certain degree of in vitro morphogenic capability, spontaneously form cysts when cultured in natural or synthetic hydrogels [START_REF] Chen | Bioengineered bile ducts recapitulate key cholangiocyte functions[END_REF], [START_REF] Funfak | Biophysical Control of Bile Duct Epithelial Morphogenesis in Natural and Synthetic Scaffolds[END_REF] and cystic organoids obtained from primary cholangiocytes have been shown to repair intrahepatic bile ducts when transplanted in a human liver undergoing ex vivo normothermic perfusion [START_REF] Sampaziotis | Cholangiocyte organoids can repair bile ducts after transplantation in the human liver[END_REF]. However, elongated duct formation in vitro in Matrigel or type 1 collagen is rarely seen [START_REF] Lewis | Complex bile duct network formation within liver decellularized extracellular matrix hydrogels[END_REF]- [START_REF] Ishida | Ductular morphogenesis and functional polarization of normal human biliary epithelial cells in three-dimensional culture[END_REF]. Another drawback is the lack of control over the direction, length, branching, and diameter of occasionally forming tubular structures.

A promising approach to building tissue constructs with controlled geometry is 3D bioprinting. In this field, Lutolf's team illustrated how extrusion-printing of a highdensity suspension of gut epithelial cells in a Matrigel/collagen hydrogel could lead to self-organization of cells into tubes with a luminal diameter of 100-200 µm [START_REF] Brassard | Recapitulating macro-scale tissue self-organization through organoid bioprinting[END_REF].

To reach an even higher resolution, in particular, to reach features in the range of 10-100 µm, digital light processing (DLP) stereolithography is particularly efficient. This approach was illustrated in a publication by Ma et al., where different types of cells (iPS-derived hepatic progenitor cells, endothelial cells, and adipose-derived stem cells) were encapsulated in imbricated 3D structures mimicking the hexagonal design of the liver lobule [START_REF] Ma | Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting[END_REF]. The resulting construct exhibited enhanced hepatic phenotypes and metabolite activity. However, the reconstruction of the biliary network was not addressed in their work.

In our study, we used normal rat cholangiocytes (NRC) and DLP stereolithography to produce Biliary Structures by PhotoPolymerization (BSPPs) with controlled geometry. After several days of culture, the self-organization of branched tubular structures guided by the predefined geometry of the hydrogel scaffold leads to the formation of BSPPs. These tubes spanning hundreds of microns had a central closed lumen with walls composed of a monolayer of cells with an epithelium phenotype, including the presence of tight junctions and primary cilia facing the lumen. Our results also demonstrate that these cholangiocyte structures possess functional transport properties of bile ducts.

Material and methods

1. Culture of cholangiocytes in 2D NRC, a cholangiocyte cell line developed by Vroman and LaRusso [START_REF] Vroman | Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells[END_REF] were maintained on collagen-coated culture vessels in Dulbecco's modified eagle medium F12 (DMEM F12, Gibco), non-essential amino acids (Gibco), lipid concentrate (Gibco), vitamin solution (Gibco), l-glutamine (Gibco), soybean trypsin inhibitor (Sigma), dexamethasone (Sigma), ethanolamine (Sigma), bovine pituitary extract (Gibco), Triiodo-L-thyronine (Sigma), Insulin-transferrin-Selenium (Gibco), and penicillin-streptomycin (Gibco) supplemented with 5% fetal bovine serum as described by De Groen et al. [START_REF] De Groen | Characterization and growth regulation of a rat intrahepatic bile duct epithelial cell line under hormonally defined, serum-free conditions[END_REF] NRCs from passages 3-12 were used for the 3D stereolithography experiments.

2.

Fabrication of BSPP using DLP stereolithography Before 3D stereolithography, photopolymerizable hydrogels were prepared with 0.2% (w/v) type I methacrylated collagen (C MA , Advanced BioMatrix), 0.3% (w/v) methacrylated hyaluronic acid (HA MA , Advanced BioMatrix), 0.14% (w/v) fibrinogen (FG, Sigma), and 1% (w/v) lithium phenyl-2,4,6 trimethyl-benzoyl phosphinate (LAP, Allevi). NRC were harvested with 0.25% trypsin-EDTA (Sigma) and diluted to cell concentrations of 10 7 NRC/mL in photopolymerizable hydrogel. The 3D stereolithography experiments were performed using an Olympus IX83inverted microscope (Olympus Corporation) equipped with a DMD-based UV patterned illumination device (PRIMO, Alveole). The wavelength of the laser is 375 nm. Rasterized image files are sent to the DMD device which is an array of approximately two million micromirrors that can be controlled individually to generate the optical pattern. The optical photopattern is then projected through a microscope lens onto the photopolymerizable hydrogel containing NRCs. Photopolymerizable hydrogels containing 10 7 NRCs/mL were dispensed by 30 µL with a pipette into the space between a methacrylated glass coverslip and a polydimethylsiloxane (PDMS) stamp. The whole device was then loaded to the microscope slide holder and exposed to the UV pattern through a 10x at 20 mJ/mm² dose. After UV exposure, the PDMS stamp was peeled off and the unpolymerized part of photopolymerizable hydrogel containing NRC was removed and washed off with PBS buffer (Sigma).

3.

Culture of BSPPs After 3D stereolithography, the BSPPs were transferred to a petri-dish or a well plate and cultured in NRC culture medium. For the Matrigel-treated BSPPs, the NRC culture medium was supplemented with 10% of Matrigel (Sigma) 2 days after photopolymerization. For all samples, culture media were renewed 3 times a week.

4.

Fluorescent dye transport assay On day 7 or day 21 of culture, BSPPs were incubated with fresh serum-free medium containing 15 μM of fluorescein diacetate (FDA, Sigma) for 5 min at 37°C and were then washed with serum-free medium three times. BSPPs were visualized using an Olympus IX83-inverted microscope with a 10x objective or Leica DMi8 microscope equipped with 10x objective and images were taken every 10 min for 1h. Temperature and CO2 concentration were kept at 37°C and 5%, respectively.

5.

Fixation, immunofluorescent staining, and confocal microscopy of BSPP To investigate the biliary epithelium formation in vitro, BSPPs cultured for a week in NRC culture medium supplemented with 10% of Matrigel for four days were fixed in 4% PFA in PBS for 15 min at room temperature, washed three times for 10 min in PBS and incubated for 1 hour in blocking solution (5% normal goat serum (Cell Signaling Technology #5425), 0,3% Triton X-100 in PBS) at room temperature. The blocking was followed by overnight incubation with the primary antibodies (1% BSA, 0,3% Triton X-100 in PBS) at +4°. After three 10 min washings in PBS at room temperature, samples were incubated with secondary antibodies (1% BSA, 0,3% Triton X-100 in PBS) for 2 hours at room temperature. Finally, samples were mounted using Aqua-Poly/Mount mounting medium and imaged using a confocal laser scanning microscope Zeiss LSM780 equipped with a 40x objective. Below is the list of used antibodies and dyes with the dilution ratios relative to stock concentrations recommended by the manufacturers: -anti-Acetyl-α-Tubulin (Cell Signaling Technology #5335) -1:800; -anti-ZO-1 (Invitrogen #33-9100) -1:100; -goat anti-Mouse IgG (Cy3) (Abcam #ab97035) -1:200; -goat anti-Rabbit IgG (Alexa Fluor 488) (Thermo Fisher Scientific #A-11034) -1:200; -phalloidin (Alexa Fluor 647) (Thermo Fisher Scientific #A22287) -1:200; -DAPI (Roche #10 236 276 001) -4 µg/ml 6.

Time-lapse image acquisition of BSPPs Time-lapse microscopy was performed at 37°C and 5% of CO2, with images taken at 30-min intervals using an Olympus IX83-inverted microscope or a Leica DMi8 microscope equipped with a 10x objective.

7.

FRAP-assays FRAP-assays were performed using a confocal microscope ZEISS LSM 780 equipped with objective W Plan-Apochromat 20x/1.0 DIC M27. Three laser wavelengths were used simultaneously for bleaching at maximum intensity: 405 nm, 458 nm, and 488 nm. The bleaching of the 7 µm-wide line across the BSPP was performed in 64 passages of the laser beam.

8.

Photopolymerizable hydrogel compositions Before 3D stereolithography, various photopolymerizable hydrogel formulations were prepared: (i) C MA /HA MA /FG with 0.2% (w/v) C MA , 0.3 % (w/v) HA MA , 0.14% (w/v) FG, and 1% (w/v) LAP (ii) C MA /HA MA with 0.2% (w/v) C MA , 0.3 % (w/v) HA MA , and 1% (w/v) LAP (iii) C MA /FG with 0.2% (w/v) C MA , 0.14 % (w/v) FG, and 1% (w/v) LAP (iv) HA MA /FG with 0.3 % (w/v) HA MA , 0.14 % (w/v) FG, and 1% (w/v) LAP (v) 0.2% (w/v) C MA and 1% (w/v) LAP (vi) HA MA with 0.3 % (w/v) HA MA and 1% (w/v) LAP (vii) FG with 0.14 % (w/v) FG and 1% (w/v) LAP. Each previously described photopolymerizable hydrogel mix was gently mixed with NRCs at a cell concentration of 10 7 NRCs/mL and were photopolymerized with a 20 mJ/mm² dose. After being incubated in NRC medium for 48 h at 37°C, the photopolymerized samples were cultured during 5 more days in NRC culture medium supplemented with 10% Matrigel. 9.

Rheological analysis

Rheological features of photopolymerized hydrogels were measured by a Discovery HR 2 rheometer (TA Instruments) equipped with a parallel plate geometry (d= 25 mm). Two different photopolymerized hydrogel formulations were prepared: C MA /HA MA /FG (0.2% C MA , 0.3 % HA MA 0.14% FG, 1% LAP) and C MA /HA MA (0.2% C MA , 0.3 % HA MA , 1% LAP). Samples with a thickness of 100 µm and 25 mm in diameter were photopolymerized with a 20 mJ/mm² dose. After being incubated in NRC medium for 24 h at 37°C, the photopolymerized samples were subjected to oscillatory measurements. In order to eliminate the environmental noise in data acquisition, the frequency sweeps were conducted at room temperature in the range of 0.1-10 Hz, and with a constant strain rate of 1%, considered to be in the linear viscoelastic range.

Results

Cholangiocytes embedded in 3D photopolymerized hydrogels of defined geometry can proliferate, migrate, and organize into branched tubular epithelial networks

Cholangiocyte cysts that form spontaneously in hydrogel environments consist of a central lumen surrounded by a monolayer of cells with apicobasal polarization [START_REF] Chen | Bioengineered bile ducts recapitulate key cholangiocyte functions[END_REF], [START_REF] Funfak | Biophysical Control of Bile Duct Epithelial Morphogenesis in Natural and Synthetic Scaffolds[END_REF], [START_REF] Lewis | Complex bile duct network formation within liver decellularized extracellular matrix hydrogels[END_REF], [START_REF] Tanimizu | Liver Progenitor Cells Develop Cholangiocyte-Type Epithelial Polarity in Three-dimensional Culture[END_REF], [START_REF] Vroman | Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells[END_REF]. When cultured in ECM derived from the decellularized liver [START_REF] Lewis | Complex bile duct network formation within liver decellularized extracellular matrix hydrogels[END_REF], some cysts randomly adopt an elongated shape. However, their size and shape cannot be controlled, and the system is inappropriate for obtaining reproducible models of functional polarized intra-lobular bile ducts. In an attempt to guide cholangiocytes' self-organization towards tubes with predetermined geometry, we used DLP stereolithography to fabricate 3D hydrogel structures of various shapes encapsulating biliary epithelial cells (Fig. 1).

After surveying a panel of ECMs that have been shown to sustain (alone or in a mixture) the culture of epithelial cells [START_REF] Freshney | Introduction[END_REF], we focused on type I collagen, hyaluronic acid, and fibrinogen. In order to print the selected hydrogels using DLP stereolithography, we screened different formulations of methacrylated collagen (C MA ), methacrylated hyaluronic acid (HA MA ), and lithium phenyl-2,4,6 trimethylbenzoyl phosphinate (LAP) -a cytocompatible photoinitiator [START_REF] Fairbanks | Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility[END_REF], with or without fibrinogen (FG) (Fig. 2-Fig. S2).

C MA is a photopolymerizable hydrogel produced from collagen that sustains good cell viability following encapsulation and retains natural cell-binding motifs [START_REF] Gaudet | Characterization of methacrylated type-I collagen as a dynamic, photoactive hydrogel[END_REF], [START_REF] Antoine | Review of Collagen I Hydrogels for Bioengineered Tissue Microenvironments: Characterization of Mechanics, Structure, and Transport[END_REF]. HA MA is also a photopolymerizable hydrogel derived from hyaluronic acid, the latter is found ubiquitously in native tissues and plays an important role in many cellular responses, such as cell signaling or cell proliferation 25-27 . FG is a component of the ECM that mediates cellular functions such as adhesion, spreading, proliferation, and migration of a variety of cell types, including fibroblasts, endothelial and epithelial cells. C MA and HA MA incorporation provides tunability of mechanical properties via variation of the UV dose and material concentration while still maintaining the essential features of the native biliary epithelial microenvironment [START_REF] Fan | Engineered 3D Polymer and Hydrogel Microenvironments for Cell Culture Applications[END_REF], [START_REF] Spearman | Tunable methacrylated hyaluronic acid-based hydrogels as scaffolds for soft tissue engineering applications[END_REF]. The incorporation of FG changes the topology of the extracellular matrix in order to provide a surface for cell adhesion, migration, and matrix remodeling [START_REF] Shepherd | Collagen-Fibrinogen Lyophilised Scaffolds for Soft Tissue Regeneration[END_REF]. As illustrated in Fig. 2A,C MA and HA MA when used alone or in combination with FG could be photopolymerized with suspended cholangiocytes. However, C MA , HA MA /FG, and C MA /FG hydrogels were rapidly degraded in the presence of living cells and thus did not offer enough stability to sustain the directed growth of any organized multicellular structure. This degradation was directly linked to the presence of living cells as the same hydrogel structures shown excellent stability over time in the absence of cells. In contrast, when all three components were mixed in a ratio of 0.2% (w/v):0.3% (w/v):0.14% (w/v) C MA /HA MA /FG, photopolymerized hydrogel structures showed substantially higher stability over time and we observed migration and reorganization of the cholangiocytes within them. This reorganization however was not observed when fibrinogen was omitted in the 0.2% (w/v):0.3% (w/v) CMA/HA MA mix.

Storage modulus (G′) and loss modulus (G″) were measured via frequencydependent oscillatory rheological analysis to investigate the viscoelastic features of the fabricated hydrogels. As depicted in Fig. 2B and Fig. S2B, both C MA /HA MA and C MA /HA MA /FG formulations exhibited a classical hydrogel feature (G′ greater than G″), where G′ and G″ were kept nearly constant in the entire frequency range (0.1-10 Hz). These measurements also showed that the C MA /HA MA/ FG mix photopolymerized at 20 mJ/mm² yielded a softer matrix compared to either C MA /HA MA photopolymerized with the same dose or C MA /HA MA /FG photopolymerized with a double dose. When NRCs were encapsulated in these three matrices, limited migration was observed for NRCs encapsulated in the slightly stiffer hydrogels (Fig. 2A, C MA /HA MA day 7 and Fig. S2A,day 7). These results suggest that it is not the presence per se of fibrinogen that is helping cell migration but rather a combination of its presence and a decrease in stiffness brought by the addition of this third component. . It is interesting to note that the storage modulus of the softer C MA /HA MA /FG photopolymerized hydrogel is in the same range of 50-800 Pa as the one measured by Lewis et al. for ECM derived from decellularized livers [START_REF] Lewis | Complex bile duct network formation within liver decellularized extracellular matrix hydrogels[END_REF] in which cholangiocytes could reorganize in vitro into cystic and elongated structures. In parallel, by optimizing UV doses and concentration of LAP, we confirmed that cells had good viability 4 hours and 24 hours after printing (Fig. S1).

We also explored various patterns of lines ranging from 10 to 500 µm in width and trees with branching angles of 30, 45, and 60° (Fig. 1 and Fig. S3). In all cases, we found that, just after photopolymerization, cells were trapped in an area larger than the one defined by the optical mask (Fig. 1C). This is most likely the result of light scattering, thereby inducing partial photopolymerization in adjacent regions and decreasing the expected resolution [START_REF] You | High-fidelity 3D printing using flashing photopolymerization[END_REF]. Nevertheless, after two days in culture, the structures had condensed (Fig. 1D) and the resulting structures were close in size to the original projected pattern, as if the cells had clustered together around the harder polymerized backbone.

Matrigel is a key component for obtaining stable BSPPs

In previous studies exploring the 3D culture of biliary epithelial cells [START_REF] Lewis | Complex bile duct network formation within liver decellularized extracellular matrix hydrogels[END_REF], [START_REF] Tanimizu | Liver Progenitor Cells Develop Cholangiocyte-Type Epithelial Polarity in Three-dimensional Culture[END_REF], type I collagen alone was insufficient to sustain the formation of any organized structure and Matrigel was required to form polarized structures with lumens [START_REF] Funfak | Biophysical Control of Bile Duct Epithelial Morphogenesis in Natural and Synthetic Scaffolds[END_REF], [START_REF] Lewis | Complex bile duct network formation within liver decellularized extracellular matrix hydrogels[END_REF], [START_REF] Tanimizu | Liver Progenitor Cells Develop Cholangiocyte-Type Epithelial Polarity in Three-dimensional Culture[END_REF]. Furthermore, the known association between biliary epithelial cells and basal lamina during bile duct morphogenesis led to the hypothesis that the basal lamina components are necessary to mimic the in vivo environment of developing bile ducts [START_REF] Tanimizu | Liver Progenitor Cells Develop Cholangiocyte-Type Epithelial Polarity in Three-dimensional Culture[END_REF]. In order to evaluate this hypothesis, we supplemented the culture media, 2 days after photopolymerization, with 10% Matrigel whose composition closely mimics the components of the basal lamina, including especially laminin-1 which is crucial for epithelial polarization of mature cholangiocytes [START_REF] Tanimizu | Liver Progenitor Cells Develop Cholangiocyte-Type Epithelial Polarity in Three-dimensional Culture[END_REF]. One week after photopolymerization, biliary epithelial cells in the samples without Matrigel failed to maintain organized structures (Fig. 3, left panel), whereas, in the presence of Matrigel, stable tubular and branched structures were maintained. The geometry of these structures correlated with the shapes on the original optical masks, with lengths up to 2 mm and widths ranging from 10 µm up to 100 µm for linear tubes (Fig. 3, right panel and Fig. S3, day 7). Cells trapped within the proximal photopolymerization zones due to light scattering were also able to actively migrate and significantly remold the photopolymerized hydrogel structures between days 2 to 7. This resulted in modification of feature shapes and sizes compared to the ones dictated by original optical masks, especially when considering complex geometries such as the smallest branches of trees (<20 µm). Seven days after photopolymerization, of which 5 days in culture media supplemented with Matrigel, we observed that the smallest branches of the 30° and 45° trees could not be resolved using our stereolithographic technique while the ones of 60° trees were often resolved (Fig. S3, day 7) and had cystic-shaped extremities.

Two weeks after fabrication, in the samples without Matrigel, BSPPs were partially degraded and detached from the glass substrate (Fig. 3 left panel, Fig. S4) whereas, in the presence of Matrigel, BSPPs could be maintained up to 21 days (Fig. 3 right panel, Fig. S3, day 14 and day 21, and Fig. S5).

These results suggest that DLP stereolithographic bioprinting can efficiently create a 3D ECM scaffold that guides proliferation, migration, and rearrangement of cholangiocytes when cultured in a medium supplemented with Matrigel and that the resulting structures can be maintained for at least 21 days after photopolymerization.

The obtained BSPPs have a monolayer wall and actively transport fluorescein from the cytoplasm into the lumen

Knowing that biliary epithelial cells spontaneously form cysts with a monolayer wall when cultured in natural or synthetic hydrogels [START_REF] Chen | Bioengineered bile ducts recapitulate key cholangiocyte functions[END_REF], [START_REF] Funfak | Biophysical Control of Bile Duct Epithelial Morphogenesis in Natural and Synthetic Scaffolds[END_REF], we sought to investigate whether the BSPPs were forming lumens and whether these constructs could elicit the basic functions of biliary epithelium.

After 7 days of culture, of which 5 days in medium supplemented with Matrigel, BSPPs were fixed, stained, and imaged using a laser scanning confocal microscope. Staining with phalloidin, anti-ZO1 antibodies, and DAPI revealed that the cholangiocytes in BSPPs are organized in a monolayer around a central lumen. These tubes could span hundreds of microns without any visible breaks in the walls. The diameter of the lumen was close to the size of the initial photopolymerized hydrogel structure as it can be seen in Fig. 4: a tube with a 20-40 µm lumen (Fig. 4A) was formed on a 50 µm photopolymerized line, while the tube with the 10-15 µm lumen (Fig. 4B) resulted from a 20 µm photopolymerized line. Moreover, F-actin distribution was characteristic of epithelial cells with dense bands along cell-cell borders. Cholangiocytes formed tight junctions as shown by ZO-1 staining suggesting the formation of an intact epithelial barrier.

We further characterized the polarization of the cholangiocytes in BSPP tubes by staining them for acetylated alpha-tubulin -a component of the primary cilia. A number of cells had developed primary cilia and these were all facing the lumen (Fig. 4C).

Functional characterization of BSPP tubes was performed using the fluorescein diacetate (FDA) assay. FDA is a non-fluorescent molecule, which living cells hydrolyze to form fluorescent fluorescein in their cytoplasm. Cholangiocytes, unlike most non-epithelial cells, can further secrete fluorescein through specific MRP2 [START_REF] Bravo | Efficient In Vitro vectorial transport of a fluorescent conjugated bile acid analogue by polarized hepatic hybrid WIF-B and WIF-B9 cells[END_REF]- [START_REF] Barth | Transcellular transport of fluorescein in hepatocyte monolayers: evidence for functional polarity of cells in culture[END_REF] transporters located on their apical membranes [START_REF] Glaser | Differential transcriptional characteristics of small and large biliary epithelial cells derived from small and large bile ducts[END_REF]- [START_REF] Luce | Chapter 4 -Pluripotent stem cell-derived cholangiocytes and cholangiocyte organoids[END_REF]. As shown in Fig. 5, our BSPPs actively transported fluorescein into the lumen, in both linear BSPPs of different diameters and branched BSPPs. The same result was obtained with Rhodamine 123 -a fluorescent compound secreted by MDR-1another anion transporter localized on the apical membrane (Fig. S7). To confirm that the fluorescent signal is coming from fluorescent molecules transported inside the luminal space and not only from the cell monolayer, we also performed a series of confocal FRAP-tests (Fig. 6) with some of the FDA-treated samples. We quenched a 7 µm-wide line across the tube and thus bleached the signal coming from the cells and the lumen. Immediately after, we observed unperturbed rapid diffusion of fluorescein inside the BSPP lumen, restoring a homogeneous signal within seconds, whereas the bleached cells remained dark. In both these last sets of experiments, the fluorescent signal appeared homogeneous throughout the branched tubular structure, confirming a closed and continuous luminal space with no gaps in the walls over hundreds of microns.

Taken together, these results demonstrate that we are able to produce geometrically controlled biliary tubes possessing structural and functional properties of intrahepatic bile ducts using DLP stereolithography to encapsulate cholangiocytes in soft hydrogels.

Discussion and Conclusions

Cholangiocytes, like many epithelial cells, are known to spontaneously form cysts, sometimes adopting an elongated tubular structure [START_REF] Chen | Bioengineered bile ducts recapitulate key cholangiocyte functions[END_REF], [START_REF] Lewis | Complex bile duct network formation within liver decellularized extracellular matrix hydrogels[END_REF]. However, the absence of control over their size and shape prevents the development of reproducible models of functional polarized intra-lobular bile ducts. In this work, we demonstrate that the control over the initial geometry of a hydrogel by DLP stereolithography can allow the guided self-organization and polarization of embedded cholangiocytes into biliary tubes with predefined geometries including linear tubes with diameters found in the intrahepatic region of the biliary tree and, most remarkably, branched networks in tree-like configurations. Hence, this work stands as a striking illustration of how engineering techniques, in particular micropatterning as well as bioprinting, can help unleash the full potential of cellular self-organization into reproducibly building functional tissues and mini-organs with higher fidelity and robustness as discussed previously in our review [START_REF] Laurent | Convergence of microengineering and cellular self-organization towards functional tissue manufacturing[END_REF] and illustrated here, as well as in our recent paper [START_REF] Gontran | Self-Organogenesis from 2D Micropatterns to 3D Biomimetic Biliary Trees[END_REF].

The composition of the gel was a critical parameter and the conjunction of all three types of matrices proved to be necessary to sustain self-organization. The precise role of each component has yet to be elucidated, although hypotheses can be made based on what is already known about these natural extracellular matrix polymers. Type I collagen is by far the most abundant protein in all vertebrates. It auto-assembles into heterofibrils with either type III or type V collagen. Heterofibrils are the structural and mechanical scaffold of most parenchymal tissues such as the lung, kidney, liver, muscle, or spleen. Type I collagen is in particular present in normal livers in the blood vessel walls and the wall of biliary ducts of the portal tracts [START_REF] Voss | Distribution of Collagen Type I and Type III and of two Collagenous Components of Basement Membranes in the Human Liver[END_REF]. C MA alone was rapidly degraded in the presence of cells in our initial experiments and the addition of HA MA improved the durability of the photopolymerized hydrogel. Indeed, hyaluronic acid is known to improve the mechanical properties of constructs in tissue engineering applications. It is also known to improve cell migration of cornea epithelial cells [START_REF] Gomes | Sodium hyaluronate (hyaluronic acid) promotes migration of human corneal epithelial cells in vitro[END_REF] and both these properties could play a role in promoting efficient and stable tissue selforganization in this study. FG in turn plays a critical role in inflammation, wound healing, and angiogenesis. Blood cells, endothelial cells, and cells of other types interact with FG while spreading into the extravascular space. FG has been implicated in particular as a substratum for epithelial cell migration during wound repair [START_REF] Donaldson | Fibrinogen-mediated epidermal cell migration: structural correlates for fibrinogen function[END_REF], [START_REF] Clark | The Molecular and Cellular Biology of Wound Repair[END_REF], [START_REF] Guadiz | Polarized secretion of fibrinogen by lung epithelial cells[END_REF]. Our results with BSPPs show that the addition of FG was indeed essential for cholangiocyte tube formation as shown in Fig. 2 where NRC structures following the initial photopattern are seen after 7 days with the C MA , HA MA , and FG mix but not with C MA or HA MA alone. However, the degree of crosslinking, affecting the stiffness of the resulting hydrogel, was also a paramount parameter since its increase for the same triad of hydrogels prevented tube formation.

As illustrated by Fig. 3 and by the supplementary movies S8 and S9, tubes form by proliferation and collective migration of cholangiocytes which seem to engulf the 3D structures, working their way between the glass and the hydrogel and eventually closing the tube when the two edges of the cell monolayers meet over the hydrogel. In some movies (in particular movies S9B and S9D) a "zipper" type of closing of these two sheets can be observed. These tubes form within a few days and Matrigel, added two days after photopolymerization, plays a critical role in the stability of these tubes over time: at early time points up to the third or fourth day after photopolymerization, depending on the initial geometry of the construct, functional tubes can be observed without Matrigel, but these are unstable and collapse rapidly (movies S8). In the presence of Matrigel, tubes are stable, functional, and can be maintained for up to 21 days. At later stages, transport assays show that these tubes dissipated the fluorescent dye very quickly from the central lumen, pointing to defects in the overall integrity of the tube after 21 days (Fig. S6). The process whereby Matrigel stabilizes these tubes has yet to be elucidated. In our conditions, the concentration of Matrigel is insufficient for actual gel formation, which means that its role is not one of physical support. We hypothesize that a component of the ECM, most probably laminin, is reinforcing the polarization of the tube by offering a basal signal to the outside of the tube.

It is interesting to note that the collapse process observed without Matrigel initiates from a central point and forms circular expanding structures that break up the initially condensed tubular structure. We hypothesize that the polarity of cells is randomly distributed in these constructs without the basal signaling brought by the laminin-containing Matrigel. It then appears that an avalanche effect leads to a collective collapse initiating from a central node. In itself, this mechanism would be interesting to elucidate further.

Finally, from an application standpoint, the microfabrication process described here provides a means to obtain functional, robust, and reproducible in vitro tissue models which are much needed for a wide range of screening applications. However, in terms of relevance for pharmacology and clinical applications, this model will need to be reproduced using cells of human origin once the reliable sources of human cholangiocytes become available. Nonetheless, our BSPPs, as well as those recently published with our collaborators using a micropatterning technique [START_REF] Gomes | Sodium hyaluronate (hyaluronic acid) promotes migration of human corneal epithelial cells in vitro[END_REF], are the first in vitro models of intrahepatic bile tubules which should prove invaluable to test receptor-mediated transport functions, drug transport and help elucidate precise mechanisms and gene functions involved in the function of the biliary tree. Indeed, the tree-like design of our biliary structures represents a planar mimic of the intrahepatic biliary network that collects bile into bigger bile ducts. We can then envision that the secreted bile in the BSPP could be collected and analyzed in an integrated microfluidic circuit, for example by fabricating the soft hydrogel structure at the extremity of a silica microcapillary (with a diameter in the range of 100µm) immobilized on the glass coverslip and subsequently allowing the cholangiocytes to self-organize into tubes, thus connecting a "soft" microfluidic network to the external "hard" microfluidic circuit. This would allow the study of the initiation of any spontaneous flow from the microfabricated biliary network and the recovery of bile, although the addition of hepatocytes to the model would then be essential to recreate the full transport cascade between hepatocytes, bile canaliculi, and cholangiocyte biliary tubules. Interestingly, in contrast to what is obtained inside bulk porous hydrogels, the tubes obtained in this setup offer full access to the outside of the tube, allowing the introduction of other cell types, such as hepatocytes and endothelial cells, at any stage of BSPP maturation. This was illustrated by our collaborators who found that in the absence of hydrogel support such as the one described here, the preseeding of endothelial cells on 2D tree micropatterns was critical to obtain stable closed cholangiocyte tubes with continuous lumens [START_REF] Gontran | Self-Organogenesis from 2D Micropatterns to 3D Biomimetic Biliary Trees[END_REF]. We now envisage the addition of hepatocytes or hepatoblasts to our model, either preorganized as spheroids, or as a cell sheet [START_REF] Matsuura | Cell sheet approach for tissue engineering and regenerative medicine[END_REF] that we would stack over the planar biliary tree. We aim to elucidate further the spontaneous reorganization of these hepatocyte/blast structures over the biliary network and study any interaction/reorganization of the bile canaliculi structures with the biliary network. The planar organization of our model and its localization in close proximity to a glass coverslip are additional advantages for conducting such studies using confocal microscopy as illustrated here.

These co-culture experiments remain however extremely challenging, notably in selecting the most promising cells, their degree of differentiation, their origin, the proper ratio between various cell types, the compatible cell co-culture media to support cell survival and migration, and in controlling proliferation. As all tissues are composed of multiple cell types, these hurdles are present in all tissue engineering endeavors and will need to be addressed as the research field moves towards more and more complex in vitro human models, (A) Cholangiocytes encapsulated in various photopolymerized 30° tree hydrogels (T30) just after photopolymerization and 7 days after photopolymerization. It is worth noting that FG is not photopolymerizable using the LAP photoinitiator. Scale bar is 200 µm. (B) Rheological analysis of the photopolymerized hydrogels used to encapsulate cholangiocytes in comparison with the storage modulus range for optimal concentration of decellularized liver ECM enabling tubulogenesis according to Lewis et al. (red framed rectangle) [START_REF] Lewis | Complex bile duct network formation within liver decellularized extracellular matrix hydrogels[END_REF]. BSPP samples were subjected to a FDA assay and imaged used a FRAP setup. A thin line (~7µm-wide) perpendicular to the long axis of the BSPP was bleached using 405 nm, 458 nm and 488 nm lasers (designated with arrowheads in the figure). Confocal imaging using a Zeiss LSM 780 equipped with a 20x objective was carried out at the indicated time points to follow fluorescein diffusion in the BSPP.

Figures

Figure 1 .

 1 Figure 1. Fabrication of BSPPs.

  Figure 1. Fabrication of BSPPs.

Figure 2 .

 2 Figure 2. In vitro characterization of various hydrogels for BSPPs.

Figure 3 .

 3 Figure 3. BSPP formation over time.

Figure 4 .

 4 Figure 4. Confocal imaging of BSPPs after 7 days of culture in media supplemented with Matrigel for 5 days.

Figure 5 .

 5 Figure 5. Functional characterization of BSPPs with an FDA assay.

Figure 6 .

 6 Figure 6. Fluorescein diffusion in BSPPs in FRAP-assays.

Figure 1 .

 1 Figure 1. Fabrication of BSPPs. (A) The scheme of DLP stereolithography. (B) Various digital patterns for BSPPs: a tree with 45° branching (T45), and lines of 100 µm (L100) and 50 µm (L50) width. (C, D) Cholangiocytes encapsulated into a correspondingly shaped photopolymerized hydrogel structures just after fabrication (C) and 2 days after the fabrication (D). Double-line scale bars are 1 mm and single line scale bars are 50 µm .

Figure 2 .

 2 Figure 2. In vitro characterization of various hydrogels for BSPPs.(A) Cholangiocytes encapsulated in various photopolymerized 30° tree hydrogels (T30) just after photopolymerization and 7 days after photopolymerization. It is worth noting that FG is not photopolymerizable using the LAP photoinitiator. Scale bar is 200 µm. (B) Rheological analysis of the photopolymerized hydrogels used to encapsulate cholangiocytes in comparison with the storage modulus range for optimal concentration of decellularized liver ECM enabling tubulogenesis according to Lewis et al. (red framed rectangle)[START_REF] Lewis | Complex bile duct network formation within liver decellularized extracellular matrix hydrogels[END_REF].

Figure 3 .

 3 Figure 3. BSPP formation over time.Two days after fabrication and culture in cholangiocyte medium, BSPPs were divided into 2 groups and cultured respectively with and without Matrigel. Cholangiocytes were encapsulated either in 50 µm photopolymerized lines (L50), 100 µm photopolymerized lines (L100), and 45° photopolymerized trees (T45) and imaged after 7 and 14 days respectively. Single line scale bar is 200 µm (50-100 µm lines) and double-line scale bar is 1 mm (45° trees).

Figure 4 .

 4 Figure 4. Confocal imaging of BSPPs after 7 days of culture in media supplemented with Matrigel for 5 days. NRC form single-layer tubular structures from an (A) 50 µm photopolymerized line and (B) tree-like branching structure. Staining for F-actin (phalloidin), tight junctions (anti-ZO-1 immunostaining), and DNA (DAPI). (C) Additional staining for acetylated α-tubulin reveals cilia facing the lumen of our tubular structures and formed by NRCs (orange -F-actin; red -ZO-1; green -acetylated α-tubulin; blue -DNA). Dashed lines indicate where the reconstructed Z-slices are located. Confocal imaging using Zeiss LSM 780 equipped with a 40x objective. Scale bar -20 µm for A and B, 5 µm for C.

Figure 5 .

 5 Figure 5. Functional characterization of BSPPs with a FDA assay. 7-day-old BSPPs were incubated with FDA and imaged with fluorescence microscope to evaluate the transport of fluorescein inside the lumen. Scale bar is 100 µm.

Figure 6 .

 6 Figure 6. Fluorescein diffusion in BSPPs in FRAP-assays.BSPP samples were subjected to a FDA assay and imaged used a FRAP setup. A thin line (~7µm-wide) perpendicular to the long axis of the BSPP was bleached using 405 nm, 458 nm and 488 nm lasers (designated with arrowheads in the figure). Confocal imaging using a Zeiss LSM 780 equipped with a 20x objective was carried out at the indicated time points to follow fluorescein diffusion in the BSPP.

We thank Didier Letourneur and Isabelle Bataille (INSERM U1148, Univ Paris 13) for access to their rheology equipment. We also thank Latifa Bouzhir for her assistance with NRC culture. We finally thank Benoit Vianay from the CytoMorphoLab (INSERM, CEA, U976) for his technical assistance with the DLP stereolithography setup.

Funding and acknowledgments

This work received the financial support of the iLite RHU program (grant ANR ANR-16-RHUS-0005).

Data availability

The raw/processed data required to reproduce these findings cannot be shared on a permanent website at this time due to technical or time limitations. The raw/processed data required to reproduce these findings can be shared by the authors upon request.

Author contributions

E.M-A. D.A. F.C. and A.F. conceived and designed the experiments; E.M-A. D.A., E.G., A.M., and W.F. performed the experimental work and contributed to the manipulation of materials, reagents, and analysis tools; E.M-A. D.A. W.F., P.D-W, F.C., J.L, and A.F. analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.


Valérie Bertrand

MD Lyvia Tiburce

MD Thibaut Sabatier

MD Damien Dufour

MD Pierre Déchelotte

PhD Marie-Pierre Tavolacci

MD Valerie Bertrand
email: valerie.bertrand@ch-havre.fr

Eater profile and associated factors in pediatric patients of the PEDIANUT cohort

Keywords: eater profile, appetite traits, associated factors, environmental factors

Appetite traits have multifactorial origins. In association with environmental and genetic factors, they could become problematic and lead to Feeding or Eating Disorders (FED). As the DSM-5 classification is not suitable for pediatric FED, another way to describe eating behavior is to distinguish the clinical profiles of "small eater" and "big eater". The aim of this study was to identify socio-demographic and medical factors associated with these profiles, and to compare problematic and non-problematic profiles. From the Pedianut study, we analysed socio-demographic, medical and family history data among 401 children according to 4 age groups (<1 year n=101, 1-6 years n=99, 6-12 years n=100, 12-18 years n=101). The information collected on eating behavior made it possible to define small eater profile (SEP) and big eater profile (BEP) using predefined grids. BEP was more frequent in adolescents (35.6%), and SEP was more frequent in children aged 1 to 6 years (34.3%). BEP was associated with having separated parents, being male and the oldest sibling (p<0.05).

Problematic BEP was associated with eating while watching television, being a girl, and having sensory disorders (p<0.05). SEP was associated, whatever age, with nonbreastfeeding, chronic illness, psychological history, sensory disorders, language delays (in the 1-6 year age group), and family history of FED (in the adolescent group) (p<0.05). This analysis of factors associated with eater profile opens new perspectives for research on risk factors associated with eating traits, which warrants further study in larger populations to delineate transition from healthy to problematic eating.

Text:

Introduction

The factors conditioning eating behaviors are not yet well understood. Appetite traits (food avoidance or food approach) could be explained by genetic processes and biological foundations (Dubois et al., 2013, Llewellyn et al., 2012, Wardle et al., 2009) but also by psychosocial factors, as parental feeding practices (Fox et al., 2020[START_REF] Jansen | Bi-directional associations between child fussy eating and parents' pressure to eat: Who influences whom?[END_REF][START_REF] Scaglioni | Factors Influencing Children's Eating Behaviours[END_REF], Russel et al., 2019[START_REF] Zhou | Appetitive Traits and Weight in Children: Evidence for Parents' Controlling Feeding Practices as Mediating Mechanisms[END_REF] and "they can be perceived as stable traits" (Guzek et al., 2020). However, in association with environmental and other genetic factors, these appetite traits could become problematic and lead to Feeding or Eating Disorders (FED), with restrictive or compulsive eating behaviors [START_REF] Herle | A longitudinal study of eating behaviours in childhood and later eating disorder behaviours and diagnoses[END_REF].

The normal development of eating profiles in children includes several steps: first, infants have reflexive sucking up to 4 months, then sucking becomes more voluntary; second complementary solid food is generally introduced between 4 and 6 months of age. Then there seems to be a sensitive period during which infants are receptive to a variety of tastes and food textures, depending on parental feeding practices, which influences eating behavior later in childhood [START_REF] Borowitz | Feeding Problems in Infants and Children: Assessment and Etiology[END_REF]. A rate of 25 to 45% of parents reported having experienced feeding difficulties with their child, but most of them resolved over time, and only 5 to 10% of children required intensive care [START_REF] Borowitz | Feeding Problems in Infants and Children: Assessment and Etiology[END_REF], Bryan-Waught et al., 2010[START_REF] Kerzner | A practical approach to classifying and managing feeding difficulties[END_REF]. As pediatric FED can differ significantly in terms of severity, DSM-5 FED definitions are not always appropriate for children (Dovey et al., 2019[START_REF] Strand | A systematic scoping review of diagnostic validity in avoidant/restrictive food intake disorder[END_REF]: in our Pedianut study (Prevalence of FED in 401 children aged between 0 and 18 years old), using the DSM-5 classification, we observed a high prevalence of "Unspecified FED" (UFED) of 9.7% 95%CI (7.2-13.0) [START_REF] Bertrand | Estimated Prevalence and Care Pathway of Feeding and Eating Disorders in a French Pediatric Population[END_REF]. As pediatric UFED are frequent, and include both restrictive and compulsive unspecified eating disorders, we used in this study a pragmatic description of pediatric eating behaviors based on appetite traits, distinguishing non-problematic and problematic "small eater profile" (SEP) and "big eater profile"(BEP).

The aim of this study was thus to investigate whether socio-demographic and/or medical factors were associated with SEP or BEP, and to compare these factors according to eating profile severity (problematic SEP or BEP i.e. FED, versus non-problematic SEP or BEP i.e. appetite traits).

Material and Methods

Study design

The Pedianut study was a non-interventional cross-sectional study conducted in one French department (Seine-Maritime, Normandy), from 3rd May 2019 to 5th March 2020. The protocol was approved by a Committee for the Protection of Persons on 18/03/2019 (N° RCB 2019-A00067-50). We analysed socio-demographic, medical and family history data among 401 children according to 4 age groups (<1 year n=101, 1-6 years n=99, 6-12 years n=100, 12-18 years n=101). Physicians used an anonymized questionnaire to conduct the interview, and parents and adolescents also completed a part of the questionnaire with age-appropriate screening tools [START_REF] Bertrand | Estimated Prevalence and Care Pathway of Feeding and Eating Disorders in a French Pediatric Population[END_REF].

Variables collected in the questionnaires.

Each questionnaire included, for all patients, demographic (age, gender) and medical data (physician measured weight, height, body mass index BMI). Overweight and underweight were assessed according to the latest validated French growth curves. After 2 years of age, children were considered underweight if BMI ≤ International Obesity Task Force (IOTF) 17 (grade II) and overweight if BMI ≥ IOTF 25 [START_REF] Bertrand | Estimated Prevalence and Care Pathway of Feeding and Eating Disorders in a French Pediatric Population[END_REF].

Questionnaires also included personal history data (prematurity, breastfeeding, digestive disorders (functional constipation, functional abdominal pain disorder, gastroesophageal reflux disease), chronic diseases, sensory disorders (tactile and taste sensitivity), surgical history, food allergies, psychiatric disorders, academic delay, language delay), lifestyle data (parents living together or not, order of birth among siblings, meals with family or not, frequency of meals watching television), family history data (eating disorders, psychiatric diseases, overweight).

Concerning dietary behavior, questionnaires included age-appropriate questions designed by the authors, and additional age-appropriate questions from screening tools extracted from the literature according to feasibility criteria (Baby Eating Behavior Questionnaire, Behavioral Pediatrics Feeding Assessment Scale, Sick Control One Fat Food, French, Eating Attitudes Test EAT-26, Three-Factor Eating Questionnaire TFEQ-R18). These tools and score results were presented in our primary study [START_REF] Bertrand | Estimated Prevalence and Care Pathway of Feeding and Eating Disorders in a French Pediatric Population[END_REF]. Physicians helped families if some questions were misunderstood.

Classification by clinical profile of small eater and big eater

After each interview, and according to the analysis of the questionnaire responses, physicians associated one or more eating behavior characteristic with each patient, according to a predefined grid, including restrictive or compulsive eating behaviors. These eating behaviors are listed in Figure 1; a patient could have one or more eating behavior item. To define eater profile, the items "tachyphagia" and "nibbling between meals" were removed (because they were subjective and not specific, and "nibbling" was present in both SEP and BEP). When children had the item "selective eating behavior", the association with other restrictive or compulsive eating behaviors led us to classify them in SEP or BEP. Thus, it was possible to compare three patient profiles (SEP, BEP, normal eater), in terms of age, BMI and associated factors.

In a second time, we made a comparative analysis of these factors according to the severity of eater profile (problematic SEP or BEP versus non-problematic SEP or BEP). Problematic eaters were defined by physicians as eaters with significant FED (selective disorder, restrictive disorder with nutritional or psycho-social consequences, phobia of swallowing and/or vomiting, emotional food avoidance, hyperphagia, compulsive disorders). These significant FED were already presented in our previous study, corresponding to 11 children with Avoidant Restrictive Food Intake Disorder (ARFID) and 35 children with UFED [START_REF] Bertrand | Estimated Prevalence and Care Pathway of Feeding and Eating Disorders in a French Pediatric Population[END_REF]. Children considered by their parents as small or big eaters without other significant restrictive or compulsive eating behaviors were considered as having nonproblematic eater profiles.

Analysis

A descriptive analysis was done for the entire cohort and for each age group. Qualitative variables were described in percentages, and continuous variables were described in median rate (minimal-maximal) for age. Chi-square was used for comparisons of categorical data.

Continuous variables were compared using the Student's t-test. Statistical analysis was done with the software XlstatBiomed 2020.3.1.

Results

The demographic (sex ratio, median age) and BMI data of the cohort, by age group, are presented in table 1. Physicians listed 427 eating behaviors among children including breastfeeding or bottle difficulties, refusal of the spoon or pieces, big or small eater, neophobic/selective eater, restrictive eater with nutritional or psycho-social consequences, phobia of swallowing and/or vomiting, emotional food avoidance, hyperphagia, tachyphagia, nibbling between meals, compulsive eating, nocturnal nibbling or hyperphagia. However, the interviews did not identify rumination, pica, anorexia, bulimia, binge eating disorder, orthorexia [START_REF] Bertrand | Estimated Prevalence and Care Pathway of Feeding and Eating Disorders in a French Pediatric Population[END_REF].

Eater profile

Prevalence by age group is presented in Figure 2. BEP was more frequent in adolescents (35.6%), and SEP was more frequent in children aged 1 to 6 years (34.3%).

Eater profile and BMI

Children with SEP were significantly more often underweight than children with BEP or normal eater profile; this finding was observed in all age groups except the 1-6 year group.

Children with BEP were significantly more often overweight than children with SEP or normal eater profile, regardless of age group (p<0.0001). When analyzing problematic eater profiles, children with problematic SEP were significantly more often underweight than children with non-problematic SEP or normal eater profile, and children with problematic BEP were significantly more often overweight than children with non-problematic BEP or normal eater profile, whatever age (p<0.05) (Table 2).

Eater profile and associated factors

SEP was associated with a history of non-breastfeeding (p=0.029), chronic illness (p=0.001), psychiatric disorders (p=0.003), presence of sensory disorders (p=0.001), food allergies related to cow's milk in the 0-1-year age group (p=0.035), language delays in the 1-6 year age group (p=0.037), and a family history of FED in the 12-18 year age group (p=0.026). BEP was associated with having separated parents (p=0.018), being the oldest sibling (p=0.007), and being male (p=0.04). Normal eater profile was weakly associated with a history of surgery (p=0.0028). (Table 3).

Factors associated with eater profile according to severity.

We identified 34 problematic SEP and 12 problematic BEP. Comparing problematic SEP and BEP with non-problematic SEP and BEP indicated some differences: problematic SEP was more frequently associated with breastfeeding, and problematic BEP with eating while watching television, being a girl, and having sensory disorders (p<0.05). (Table 4)

Discussion

This study provides additional data on factors associated with eating behavior in pediatric patients, notably, language delay in small eater profile, and sensory disorders in problematic big eater profile.

4.1 Factors associated with small eater profile. We found an association between language delay and SEP in the 1-6 year age group. This association has been rarely studied in the literature, while the development of language and orality are probably interconnected. Only one study reported an association between language delay, chewing and selective eating disorders [START_REF] Palladino | Language and eating problems in children: co-occurrences or coincidences?[END_REF]. In a more recent study, authors suggested an association between children born prematurely who had initial feeding disorders and language delay [START_REF] Adams-Chapman | Association between feeding difficulties and language delay in preterm infants using Bayley Scales of Infant Development-Third Edition[END_REF]. In our study, no children with language delay in the 1-6 year age group had a history of grommets for otitis media, nor a history of tongue tie whose contribution to eating and language disorders remains controversial (O'Shea JE et al.,2017[START_REF] Wang | The effect of ankyloglossia and tongue-tie division on speech articulation: A systematic review[END_REF]. Concerning academic delay, this tended to be more frequent in all ages of our problematic SEP group (p=0.05), which includes restrictive UFED and ARFID, but there are still no data on academic level in literature for these two categories. This contrasts in literature with high academic level in children with anorexia nervosa [START_REF] Sundquist | School Achievement and Risk of Eating Disorders in a Swedish National Cohort[END_REF].

In our study, SEP was also associated with non-breastfeeding, sensory disorders, chronic illness, psychological disorders, which have already been cited in the literature with restrictive FED. There are indeed many factors that can lead a child to associate discomfort rather than enjoyment with eating. Restrictive feeding disorders have also been associated with gastroesophageal reflux, chronic abdominal pain, constipation, prolonged hospitalization, surgery, prematurity, food allergy, lack of weaning, abnormal sensory environment, tube feeding or gastrostomy, bad positioning at mealtimes, inappropriate mealtime frequency/duration, and parents' psychological fragility/anxiety [START_REF] Benjasuwantep | Feeding problems in healthy young children: prevalence, related factors and feeding practices[END_REF][START_REF] Borowitz | Feeding Problems in Infants and Children: Assessment and Etiology[END_REF][START_REF] Chao | Association of Picky Eating with Growth, Nutritional Status, Development, Physical Activity, and Health in Preschool Children[END_REF], Green et al., 2015[START_REF] Johnson | Eating difficulties in children born late and moderately preterm at 2 y of age: a prospective population-based cohort study[END_REF][START_REF] Kerzner | A practical approach to classifying and managing feeding difficulties[END_REF], Lapillone et al., 2019, Silvia-Garcia et al., 2016). Breastfeeding and a higher level of parental education seem to facilitate a normal eater profile (Harris et al., 2016[START_REF] Iron-Segev | Associations of breastfeeding with bulimic behaviors and eating disorders among adolescents[END_REF], while highly controlling or negligent parental feeding practices are associated with poorer child appetite regulation, and with restrictive (or compulsive) behavior (Silvia [START_REF] Garcia | Latina mothers' influences on child appetite regulation[END_REF][START_REF] Zhou | Appetitive Traits and Weight in Children: Evidence for Parents' Controlling Feeding Practices as Mediating Mechanisms[END_REF]. Sensory disorders are described in restrictive and selective behaviors (Dovey et al., 2019[START_REF] Zickgraf | Journal of clinical child and adolescent psychology : the official journal for the Society of Clinical Child and Adolescent Psychology[END_REF]. Chronic diseases requiring specific diets (food allergies, cystic fibrosis, type 1 diabetes, inflammatory bowel disease, irritable bowel syndrome) or having anatomical abnormalities like esophageal atresia, could induce or co-exist with restrictive or selective behaviors [START_REF] Avila | Eating disorders in adolescents with chronic gastrointestinal and endocrine diseases[END_REF][START_REF] Borowitz | Feeding Problems in Infants and Children: Assessment and Etiology[END_REF][START_REF] Conviser | Are children with chronic illnesses requiring dietary therapy at risk for disordered eating or eating disorders? A systematic review[END_REF], Menzies et al., 2017[START_REF] Quick | Chronic illness and disordered eating: a discussion of the literature[END_REF][START_REF] Reed-Knight | Adolescents with irritable bowel syndrome report increased eating-associated symptoms, changes in dietary composition, and altered eating behaviors: a pilot comparison study to healthy adolescents[END_REF][START_REF] Wróblewska | Increased prevalence of eating disorders as a biopsychosocial implication of food allergy[END_REF]. For some authors, being the oldest child, eating in front of screens or not eating with the family are deleterious, but this was not associated with problematic or non-problematic SEP in our study [START_REF] Benjasuwantep | Feeding problems in healthy young children: prevalence, related factors and feeding practices[END_REF], Goday et al., 2019, Green et al., 2015[START_REF] Kerzner | A practical approach to classifying and managing feeding difficulties[END_REF].

Psychological disorders were associated with SEP in this study: there are no data in literature about this factor and no problematic restrictive appetite trait, but children with FED frequently have depression or anxiety disorders [START_REF] Herpertz-Dahlmann | Children in Need-Diagnostics, Epidemiology, Treatment and Outcome of Early Onset Anorexia Nervosa[END_REF], Mohammadi et al., 2020), and selective/restrictive eating behaviors or ARFID are also associated with attention deficit hyperactivity disorder and autism spectrum disorders [START_REF] Cooney | Clinical and psychological features of children and adolescents diagnosed with avoidant/restrictive food intake disorder in a pediatric tertiary care eating disorder program: a descriptive study[END_REF], Eddy et al., 2015, Fisher et al., 2014[START_REF] Kambanis | Prevalence and correlates of psychiatric comorbidities in children and adolescents with full and subthreshold avoidant/restrictive food intake disorder[END_REF], Lucarelli et al., 2017[START_REF] Nicely | Prevalence and characteristics of avoidant/restrictive food intake disorder in a cohort of young patients in day treatment for eating disorders[END_REF][START_REF] Norris | Exploring avoidant/restrictive food intake disorder in eating disordered patients: a descriptive study[END_REF][START_REF] Sharp | Feeding problems and nutrient intake in children with autism spectrum disorders: a meta-analysis and comprehensive review of the literature[END_REF]. In the adolescent age group, we found an association between SEP and a family history of eating disorders. ARFID, as anorexia nervosa, have been described with a family history of anxiety and depression and/or eating disorders [START_REF] Bion | Early restrictive feeding disorders: Quantitative assessment of parent/infant feeding interactions[END_REF][START_REF] Bryant-Waugh | Feeding and eating disorders in childhood[END_REF], Green et al., 2015, Lucarelli et al., 2018, Martini et al., 2019[START_REF] Nicholls | Childhood eating disorders: British national surveillance study[END_REF][START_REF] Norris | Exploring avoidant/restrictive food intake disorder in eating disordered patients: a descriptive study[END_REF], and food selectivity could be related to genetic background (Elkins et al., 2018[START_REF] Knaapila | Food neophobia shows heritable variation in humans[END_REF].

Factors associated with big eater profile. BEP was associated in the present study with

being male, the oldest sibling, and having separated parents. A biopsychosocial model explaining sex differences in eating behaviors has been proposed [START_REF] Keller | A Biopsychosocial Model of Sex Differences in Children's Eating Behaviors[END_REF], arguing that "a feminine identity is characterized by eating smaller portions, consuming less meat, and preferring healthier options to maintain appearance, while a masculine eating identity is characterized by feeling full, with a focus on physical performance" and that "social influences include differences in parental feeding practices directed at males and females", as boys are generally encouraged to eat more than girls (Loth et al., 2013), while society encourage girls to be thin. Adolescent girls may be more sensitive to internal satiety cues and boys have higher scores for food responsiveness (de la Fuente-Reynoso et al., 2020[START_REF] Hunot-Alexander | Confirmation of the Factor Structure and Reliability of the 'Adult Eating Behavior Questionnaire' in an Adolescent Sample[END_REF]. However, recent French studies found no significant gender difference in children in terms of BMI [START_REF] Verdot | Children and adults body mass index in France in 2015. Results of the Esteban study and trends since 2006[END_REF]. Some studies have analysed birth order among siblings, one study found an association between higher caloric intake diet with having two or more older siblings, possibly because parents with larger families have less time to cook healthy food, but there are few data about the oldest sibling [START_REF] Coxon | Sources and Determinants of Discretionary Food Intake in a Cohort of Australian Children Aged 12-14 Months[END_REF], Lioret et al., 2015). Having separated parents particularly before 6 years of age was associated with a higher long-term BMI in one study, but this may be related to psycho-economic-social biases (Goisis et al., 2019). Whether parental divorce is a risk factor for eating disorders remains debated [START_REF] Bachar | Surgery and parental separation as potential risk factors for abnormal eating attitudes-longitudinal study[END_REF], O'Connor et al., 2016).

Although we did not highlight this factor, breastfeeding may protect children and adolescents against overweight through a better acceptance of vegetables [START_REF] Specht | Duration of exclusive breastfeeding may be related to eating behaviour and dietary intake in obesity prone normal weight young children[END_REF][START_REF] Wang | BMI growth trajectory from ages 2 to 6 years and its association with maternal obesity, diabetes during pregnancy, gestational weight gain, and breastfeeding[END_REF], but not from eating disorders like bulimia [START_REF] Iron-Segev | Associations of breastfeeding with bulimic behaviors and eating disorders among adolescents[END_REF]). An authoritative parenting style could limit children's intake of snack food and lower the risk of obesity, while parental emotional feeding and parental restrictive feeding favor children's overeating and high BMI [START_REF] Jansen | Extending the validity of the Feeding Practices and Structure Questionnaire[END_REF][START_REF] Rodgers | Maternal feeding practices predict weight gain and obesogenic eating behaviors in young children: a prospective study[END_REF][START_REF] Scaglioni | Factors Influencing Children's Eating Behaviours[END_REF][START_REF] Zhou | Appetitive Traits and Weight in Children: Evidence for Parents' Controlling Feeding Practices as Mediating Mechanisms[END_REF].

We did not study this factor, but a low socioeconomic status has also been associated with being overweight [START_REF] Scaglioni | Factors Influencing Children's Eating Behaviours[END_REF][START_REF] Shrewsbury | Socioeconomic status and adiposity in childhood: a systematic review of cross-sectional studies 1990-2005[END_REF], and it is considered that parents with high socioeconomic status have better resources to buy food, better knowledge about healthy food and good feeding practices (Harris et al., 2016, Saxton et al., 2009).

Although a family history of being overweight and psychological disorders were not significant factors in our cohort, it is known that genetics is an important risk factor for obesity, including compulsive eating disorders [START_REF] Lamerz | Prevalence of obesity, binge eating, and night eating in a cross-sectional field survey of 6-year-old children and their parents in a German urban population[END_REF], Lee et al., 2018), and that obese adolescents with compulsive disorders have more anxiety and depression disorders [START_REF] Rancourt | Overlap in Eating Disorders and Obesity in Adolescence[END_REF].

4.3

We identified some differences between problematic SEP or BEP and nonproblematic SEP or BEP, but these findings should be interpreted with caution because of our limited population. Our finding that children with problematic SEP were more frequently breastfed is at variance with previous findings supporting a protective role of breastfeeding against restrictive eating disorders ( Harris et al., 2016[START_REF] Iron-Segev | Associations of breastfeeding with bulimic behaviors and eating disorders among adolescents[END_REF]. Our observation that problematic BEP was associated with eating in front of screens is in agreement with previous reports binding overweight in adolescents with screen viewing, low family economic status, high food intakes of a low-quality diet with sugar-sweetened beverages and high sugar and high fat foods [START_REF] Avery | Associations between children's diet quality and watching television during meal or snack consumption: A systematic review[END_REF][START_REF] Robinson | Screen Media Exposure and Obesity in Children and Adolescents[END_REF][START_REF] Scaglioni | Factors Influencing Children's Eating Behaviours[END_REF]. In contrast, eating with the family during adolescence could favor healthy diet habits in young adulthood [START_REF] Harrison | Systematic review of the effects of family meal frequency on psychosocial outcomes in youth[END_REF]; however, we were not able to distinguish eater profiles according to this factor in the present study, which may be due to the insufficient number of patients in the adolescent group. Problematic BEP was also associated with being a girl, which could be explained by the fact that adolescent girls show higher levels of emotional over-eating than boys [START_REF] Hunot-Alexander | Confirmation of the Factor Structure and Reliability of the 'Adult Eating Behavior Questionnaire' in an Adolescent Sample[END_REF][START_REF] Keller | A Biopsychosocial Model of Sex Differences in Children's Eating Behaviors[END_REF].

Interestingly, problematic BEP was associated with having sensory disorders (tactile and taste), which is a factor rarely screened in studies. Navarette-Muñoz et al reported recently a significant association between an increase in BMI and a higher prevalence of atypical tactile sensivity in a general population of children aged 3-7 years, possibly due to a poorer quality of diet induced by selective eating behaviors (Navarette-Muñoz et al., 2020). Sensory avoiding behaviors associated with higher BMI have also been described in children with autism spectrum disorder, with a high prevalence of obesity, and authors hypothesized that these sensory avoiding behaviors induce both less physical activity and higher selective eating behaviors (Lawson et al., 2016).

4.4

Concerning eater profile and BMI, SEP and BEP were associated respectively with being underweight and overweight. This fact was already observed in previous pediatric or adult studies, with a good correlation between appetitive traits and BMI (Aschroft et al., 2008, Hunot-Alexander et al., 2016, Mallan et al., 2017[START_REF] Van Jaarsveld | Prospective associations between appetitive traits and weight gain in infancy[END_REF][START_REF] Webber | Eating behaviour and weight in children[END_REF].

Strength and limitations of the study.

Our study is one of the rare pediatric studies based on clinical interviews conducted in a general population. But we cannot exclude the fact that parents may have misperceived their child's eating behavior, and adolescents may have minimized their eating behaviors, which can induce an initial classification bias. In addition, some factors as chronic diseases, history of surgery, food allergies may be different in terms of severity. Lastly, as this was an exploratory study, with no a priori hypothesis on statistical power, correction for multiple testing was not performed. Therefore, caution is needed in the interpretation of significant results which will need to be confirmed in further studies.

Conclusion

This analysis of factors associated with eater profile opens new perspectives for research on risk factors associated with eating traits in children. We found two new factors rarely screened in literature: language delay in small eater profile, and sensory disorders in problematic big eater profile. These preliminary results need to be confirmed with prospective studies conducted in larger cohorts. A better knowledge of the psycho-social and environmental factors that could make appetite traits become problematic eating behaviors could help improve the prevention of these restrictive or compulsive disorders.

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Classification of patients: normal eater profile, small eater profile (SEP), or big eater profile (BEP).

Analysis of socio-demographic and/or medical factors associated with SEP or BEP, Comparative analysis of these factors according to the severity of eater profile (problematic SEP or BEP versus non-problematic SEP or BEP).

Attribution of one or more eating behavior characteristic for each patient

Restrictive eating behaviors

Figure 1 :

 1 Figure 1: Flowchart showing the strategy for classifying the clinical profile of small eater or big eater.

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Table 1 :

 1 Demographic data and BMI data of the pediatric population, by age group. Distribution of eater profile by age group.

		Compulsive eating behaviors	Others
	Breastfeeding difficulties,	Big eater,	Pica
	Difficulty taking the bottle,	Hyperphagia,	Rumination
	Refusal of the spoon,	Tachyphagia,	Orthorexia
	Refusal of the pieces,	Nibbling between meals
	Small eater,	Compulsive eating,
	Nibbling between meals	Selective eater and big/compulsive eater
	Neophobic/selective eater,	Nocturnal nibbling,
	Restrictive eater with nutritional or psycho-	Nocturnal hyperphagia,
	social consequences,	Bulimia nervosa,
	Phobia of swallowing and/or vomiting,	Binge eating disorder
	Emotional food avoidance,
	Anorexia nervosa

Table 3 :

 3 Associated factors according to eater profile and age (results/number of answers in %).

	n=401	Big eater profile	Small eater profile	Normal eater profile	p
		n=100 (%)	n=80 (%)	n=221 (%)
	Prematurity	4/98 (4.08%)	7/80 (8.75%)	18/213 (8.45%)	P=0.34
	0-1year
	1-6 years
	6-12 years
	12-18 years

Acknowledgements

The authors are grateful to Nikki Sabourin-Gibbs, Rouen University Hospital, for her help in editing the manuscript.