Ludovic Broche

Bertrand Favier

Hélène Rougé-Labriet

Sabine Drevet

Bernard Lardy

Emmanuel Brun

Benjamin Lemasson

Calcified cartilage revealed in whole joint by X-ray phase contrast imaging

Keywords:

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

Introduction

Osteoarthritis (OA) represents the most widespread osteoarticular disease. It constitutes a growing public health problem, with our aging population and expanding obesity wave. Nowadays it represents the most common cause for disability with consecutive annual health care costs of up to 128 billion dollars in the US 1,[START_REF] Hawker | Osteoarthritis is a serious disease[END_REF] . New therapeutic and symptomatic options for cartilage diseases have been tested in the past years in animal models and clinical, such as analgesic treatment . Indeed, MRI cannot so far reach the few microns spatial resolution required to characterize properly microcalcifications. Fluorescence imaging is also used to quantify the distribution of specific chemical elements such as reactive oxygen species but is limited to imaging at the macro scale and in two dimensions [START_REF] Xie | Quantitative imaging of cartilage and bone morphology, reactive oxygen species, and vascularization in a rodent model of osteoarthritis[END_REF] Using a wave approach with X-rays, the refraction index of light element materials can be a thousand times greater than its counterpart the absorption factor for the wavelength in radiology [START_REF] Bravin | X-ray phase-contrast imaging: from pre-clinical applications towards clinics[END_REF] . This induces a much greater contrast for soft tissues with X-ray imaging methods based on the detection of the light refraction or with a wave description the phase . Nevertheless, to our knowledge, only a few studies evaluated PCI for to study small animal models albeit being a fast 3D imaging technique, contrast agent independent, in-vivo compatible and with a high special resolution.

The objectives of this study are to present the very first results obtained using PCI on a murine OA model and to evaluate the PCI capability to depict soft and hard tissues of the knee compared to the established preclinical imaging modalities. We also propose several new 3D morphometric surrogate markers with a special focus on articular cartilage, calcified cartilage and osteophytes.

Methods

Overall Protocol

All imaging protocols were done post-mortem on six mice, in which we analyzed both knees (n control = 6, n MIA = 6). The removed knees were formalin fixed, agarose imbedded, and synchrotron phase contrast imaging (PCI) was performed, followed by MRI and conventional x-ray microscopy. Then samples were then sliced for histological analysis.

Animal Manipulation

In accordance with the Directive 2010/63/EU, the experiments were performed in an agreed animal facility (C3851610006) evaluated by an Ethical Committee for Animal Welfare and authorized (APAFIS #13792-201802261434542 v3). OA pain model was induced by bilateral single intra-articular injection of monoiodoacetate (MIA-OA, Sigma, Saint Quentin-Fallavier, France) in the knee. Six 6 weeks old C57BL6 female mice (Janvier Labs, France) were anaesthetized by xylazine and ketamine injection (10 and 100 mg/kg), a 0.5 mm skin incision was made over both knee, MIA (0.1 mg/μl, 5μl) or the same volume of phosphate buffer (PBS) for control animals was injected through the infra-patellar tendon perpendicular to the tibia using Hamilton syringe with 25μL capacity and dispensable 0.4 mm diameter needles. Mice received buprenorphine once (0.1mg/kg, sub-cutaneous) for pain management during recovery. Mice were monitored for 10 weeks for obvious locomotor disability by visual observation and Rotarod testing (Bioseb, Vitrolles, France) and then sacrificed. Knees were removed, fixated in 4% formaldehyde for 48h and embedded in PBS with 2 % agarose.

Synchrotron X-ray Imaging

Image acquisition was performed at the biomedical beamline of the ESRF (Grenoble, France). A propagation-based PCI technique was used to enhance contrast in between small density changes by capitalizing on both x-ray absorption and refraction. This imaging technique uses highly coherent radiation, with a long sample to detector distance (11 m) to measure interferences between refracted radiations (see Figure 6). Synchrotron X-ray sources are particularly suited to provide such characteristics, allowing for an acquisition time of 8 min at an isotropic voxel size of 6.1×6.1×6.1μm 3 . A wiggler source was used to produce broad-spectrum synchrotron radiation. The continuous spectrum was filtered to a 52 keV narrow energy band by a double bent Silicium crystal monochromator. This relatively high energy was chosen as a compromise between contrast, loss of spatial resolution due to streak artefacts caused by phase shifts bigger than a pixel and the very long distance imposed by the beamline configuration at the time of the experiment. Images were recorded with a sCMos camera (PCO edge) with a 2560 × 2160 pixels chip. The projections acquired 11 m away from the sample were brought into focus using the Paganin algorithm 33 , the ratio was set to 1000 (the theoretical value of bone) knowing the setup geometry and the approximate elemental composition of the sample. A total of 2500 projections was acquired to cover the 360 degree.

Magnetic Resonance Imaging

MRI was performed at 9.4T in a horizontal bore magnet (Bruker Biospec 94 / 20 Avance III-HD, Bruker BioSpec, Ettlingen, Germany) at the IRMaGe MRI facility (La Tronche, France) equipped with an helium cooled cryoprobe and a phased-array surface receive coils configuration. A 3D ultra-short echo time (UTE)

Conventional X-ray microtomography

Samples were imaged on a conventional microtomograph (EasyTom XL from RXSolution, Chavanod, France) at the SIMaP-GPM2 lab (Grenoble, France). A conventional microfocus X-ray tube was used, with a maximum high voltage set to 60 kVp coupled with a 127 μm flat panel detector allowing for an acquisition time of 11 min for a 11.1 × 11.1 × 8.8 mm 3 volume at a voxel size of 6.06μm 3 . The source spot size was adjusted to be inferior to 5µm. The number of projections was 3600.

Histological Analysis

Samples were paraffin embedded; 4μm slices were cut every 30μm and colored using the Safranin-O Fast-Green technique described by Glasson 17 except that the optimal decalcification time was 48h in 20 % EDTA. Histological images were acquired with a Zeiss Axio Scan slide scanner. The OARSI histological grading system guideline was used to grade the severity of OA 17 . This semi-quantitative scoring system ranged from 0 to 6 (the higher the more severe) and was applied to all four quadrants (score out of 24): medial femur, medial tibia, lateral femur, lateral tibia.

PCI Processing and Analysis

Bone and calcified cartilage structure were segmented using a region-growing algorithm 34 . A morphological opening was then used to remove structures smaller than 3 voxels in diameter [START_REF] Asano | Optimization of gray scale morphological opening for noise removal in texture images[END_REF] . All segmented elements (bone and calcified cartilage), not connected to the tibia, femur, fibula, menisci and patella were labelled as calcifications, for which we reported their number and volumes. The marching cube algorithm 36 was applied to every segmented structure to generate the polygonal surface meshes. A feature preserving mesh smoothing 37 algorithm was then applied to remove aliasing artifacts. Distance map was calculated from the tibia and femur segmented mesh with a fast-marching algorithm 38 . From the distance map, the surface of maximum distances (i.e. surface for which the tibia and femur are equidistant) was extracted to compute the mean and standard deviation of the interspace. Effective thickness of the dense cartilage structures was characterized for both the tibia and femur using a maximal included ball algorithm through the iMorph software 39 . Using the same regions of interest at the condyle positions, the segmented calcified cartilage where numerically filled with growing spheres to fill up the entire volume. The distribution of all the spheres diameters was then normalized to their total numbers (n sphere ≈ 300). All the curvature point distribution were recorded with negative value for concave surfaces and positive value for convex surfaces roughness was computed with the VTK library 40 .

Statistics

Pairwise comparisons were performed by using independent Student-Newman-Keuls tests on each condyle interspace with the tibia, in between experimental conditions. When both condyles were included in one tested group a Kolmogorov-Smirnov test was applied to consider the distributions bimodality.

Asymmetric aperture map distributions were pairwise tested in between conditions using a Man-Whitney test. Finally, a Levene's test was used to test pairwise comparison of the curvature map standard deviations. All statistical tests were carried out with a significance level of p < 0.01 using the SciPy statistical library with the Python3.7 language 41,42 .

Results

Comparison of the Phase Contrast Imaging technique to MRI, X-ray micro-tomography

Figure 1 shows a representative slice, extracted from each 3D volumes, of a MIA-OA mice knee. MRI (Figure 1A) with a ultra-short echo time (UTE) sequence shows a readable contrast between bone and tissue structure as well as within the tissue structures (tendons, muscles, cartilages). But, the lack of image definition (pixel size of 50μm Conventional X-ray micro-tomography (Figure 1B) with its high resolution shows a precise morphology of the bone structure but completely lacks contrast within soft and bony tissues. PCI (Figure1C) allies a high image precision good differentiation between all anatomical elements of the knee joint (muscle, tendon, calcified cartilage, articular cartilage, bone and calcifications). The insets shown in Figure 1D andE are situated in the joint space and show the same zone with adapted gray scales for enhancing either the articular cartilage and synovial fluid (Figure 1D) or the calcified cartilage (Figure 1E). On these example, one can clearly see, in the same imaging modality, all the element of the knee joint: cartilage, calcified cartilage and chondrocytes. All the imaging dataset for the leg imaged with the different modalities are available at doi://10.17605/OSF.IO/W4P6M.

Validation of PCI results with histological images

To validate our PCI data with the gold standard histology, we present in Figure 2, a comparison on a representative slice of a control (Figure 2A,C,E,G,H) and a MIA-OA (Figure2B,D,F,I,J) mice knees acquired by PCI (Figure2A-D) and safranin-0 colored histological sections (Figure2E-J) with a focus on the medial femoral condyle area (C,D,G-J). To highlight the unique capabilities of PCI to render such contrast, the PCI contrast window was set to highlight the density changes within the calcified structures. The articular cartilage is therefore too dark to be visible and we indicate by a red dotted line its boundaries found thanks to a segmentation based on another contrast windowing (see method section). The distinction between the subchondral bone ( †) and the calcified cartilage ( * ) can be visualized with PCI on both the control and the MIA slices. Dark rounded dots, visible within the calcified cartilage in PCI are ascribed to chondrocytes (γ). In the knee histological sections, the purple safranin-O stains the cartilage peptidoglycans. The calcified cartilage can be found on these images by a reddish color that seem to be correlated with the PCI images in both the control and MIA images. In the MIA-treated knee, osteoarthritis is assessed by disorganization and broadening of the cartilage, safranin-O negative areas at the surface (Figure 2I), clefts at the cartilage surface with dead cell ghosts (Figure2J). These features can also be seen on the PCI slices, with a thicker calcified cartilage layer and with darker and larger chondrocytes.

3D histomorphometry analysis of two joints

Figure 3 and Figure 4 show representative examples of the different surrogate markers PCI may provide on both the control and MIA knee joints. After the segmentation of our images in bone and calcified cartilages zones we can measure virtual 3D histomorphometry quantities that are non-accessible with gold standard 2D histology.

The Figure 3 presents rapid and direct 3D measurements of features similar to those used in human X-ray imaging or in histological evaluation of OA. We calculated on our segmented bone structures the mean femur to tibia inter-space at the condyle positions in three dimensions (Figure 3A). The regions of interest where the mean distance and their standard deviation were calculated are indicated by the gray square. It is interesting to note that by averaging the mean inter-distance of both condyles no statistical differences appears in between the Control and the MIA joints (56.7±8.5μm vs 57.4±17.3μm) (Plain color bars in Figure 3D ). But the MIA-OA knee displays a destabilization between his two condyles with a thin medial femur/tibia interspace (39.4±7.1μm) and a large lateral femur/tibia interspace (70.3±6.5μm), while such asymmetry is not present on the control knee (54.2±8.5μm vs 58.2±8.5μm ).

Thanks to a granulometry algorithm [START_REF] Brun | Fast granulometry operator for the 3d identification of cell structures[END_REF] , we can calculate in three dimensions the local thickness of the different tissues. The Figure 3B shows on a sagittal cross-section the distribution of the local thickness whilst the Figure 3C represents those values projected in 3D on the segmented surfaces. The full distribution of calcified cartilage thickness in both femur and tibia for each condition is shown in the lower panel E and F. The thickness analysis on the calcified cartilage shows an overall increased in thickness with the MIA-OA knee 46.9±22.4μm compared to the control 23.4±17.2μm thickness. Bone wise, the femur and tibia calcified cartilage mean thickness are 47.6 ± 15.7 μm and 46.3 ± 22.4 μm respectively on the MIA-OA knee, and 28.7 ± 18.6 μm and 18.1 ± 13.9 μm respectively in the control knee. No statistical differences were found in between the femur and tibia on each of the two conditions for this mouse. We can nevertheless note that the local thickness is the largest in the MIA joints within the condyles where the femur/tibia interspace is the smallest. The total volume of MIA calcified cartilage in the full join is also largely superior with V MIA = 9.42 × 10 ).

Figure 4I-L shows the 3D distribution of all the segmented osteophytes in both control (I) and MIA (J)

where the osteophytes were colored. Osteophytes were defined as high density elements not connected to the three main bones or the menisci. The graph in Figure 4K and L, shows a 2D projections in sagittal and transverse coordinates where the volume of the osteophytes is schematized by a circle with the same volume. As one can see, the control knee presents some osteophytes probably due to the injection of PBS but the MIA-OA knee displays a significantly higher amount (n OA = 31 vs n Ctrl = 13 ) with a larger total volume (V OA = 4.29 × 10 2 μL vs V Ctrl = 6.01 × 10 3 μL).

Quantification of the whole image database

All the computed metric results are recapitulated in table1. Figure5 displays the comparison of the morphological parameters computed in the same fashion of the above examples on the PCI images but for the all the MIA injected legs (n = 6) and their controlateral legs (n = 6). Figure 5A presents the mean interspace distance between femur and tibia taking into account both condyles (bars in plain color). The distance is significantly higher (p-value ≤ 0.01) for the control than for the MIA knees. The group displays the same trend as in the above example when measuring specifically the interspace in between condyles.

The same strong destabilization appears in between the MIA medial and lateral condyle (25.6±6.8μm vs 70.7±4.3μm) while in the control group such effect is statistically non relevant (55.6±9.1μm vs 56.7±4.1μm).

The same trend can be observed when measuring the calcified cartilage thickness (Figure 5B andC). The MIA model animals display a significant higher thickness compared to the control for both the femurs (24.2±7.3μm vs 47.7±15.4μm) and the tibia (25.1±8.1μm vs 44.4±17.2μm).

The surfaces roughness at the calcified and articular cartilage interface shows a smooth surface along the control and the MIA group for both the femur and tibia (Figure 5D andF). On the other hand, as shown in

Discussion

The main objective of this work was to study whether Phase Contrast Imaging is a suitable imaging modality for studying at high resolution a murine osteoarthritis model. We demonstrated that compared to conventional MRI and conventional micro CT images, PCI technique has a higher image sensibility and resolution. It allows in a single scan a full representation of the soft and calcified tissue structures at the same resolution than conventional micro-CT that is limited to bone visualization only. We showed for the first time the possibility to distinguish in 3D, using a non-invasive technique i.e. in complete unaltered mice knees, the articular and calcified cartilages as well as the subchondral bone and some clustered chondrocytes.

In this study, for sake of comparison with literature, we used the most characterized chemical-induced OA mouse model based on the injection into the rodent knee joint of mono-iodoacetate (MIA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, a glycolysis enzyme , we also found a drastic increase in micro-calcification number and volume. Such metrics could potentially be a relevant biomarker to spot early stage of MIA-OA model. On the other hand, surface roughness of the subchondral bone and calcified cartilage tidemarks did not exhibit any pertinent outcomes on that model. We also observed no sign of subchondral bone alteration such as osteophytes, vascular infiltration or osteoclasts. In a hypothetical clinical usage of the PCI technique for osteoarthritic joint detection, calcified cartilage thickening could potentially join the already existing diagnostic radiographic hallmarks.

The secondary objective of this study was to present some surrogate markers this 3D imaging modality may offer. Besides the measurements of features classically admitted as hallmarks of OA (cartilage thickness, distance between bones), we proposed new morphological biomarkers to grade cartilage alterations, calculated here in 3D. These markers were calculated on the calcified cartilage due to the lack of articular cartilage on some animals of the MIA group, but in principles all these morphological parameters could have been measured on other tissues. In summary, we have shown that synchrotron radiation phase contrast imaging is a promising technique to rapidly detect and quantify microstructures for the investigation of small animal models of osteoarthritis. For example, the mean distance between the tibial plate and the femur is not different between the control and the MIA group but a difference can be observed if one measure separately the condyles. As expected, the calcified cartilage is thicker than in the OA group even in a OA low grade knee. The number and volume of calcifications seem to be a good indicator of the disease. The roughness of the interface between the calcified cartilage and the subchondral bone seem to be discriminant as well contrary to the interface between articular and calcified cartilage.

The main limitation of this study is the low number of investigated samples (n=6). Future studies will be carried out in larger groups of animals and other mouse model (surgically induced for example) to further evaluate the usefulness of PCI discriminant markers in the OA diseases. This limitation is mainly due to the limited access to synchrotron. But as previously shown the PCI technique is perfectly compatible with the preclinical in-vivo applications [START_REF] Bech | In-vivo dark-field and phase-contrast x-ray imaging[END_REF][START_REF] Fardin | Enhancing lung tumor visibility using in-vivo analyzer-based x-ray phase contrast imaging in mouse: A feasibility study[END_REF] . The next steps will consist in performing such experiment in-vivo on laboratory systems at a reduced radiation dose. Indeed synchrotron limited access, does not allow to follow-up the pathologies or at the cost of using many animals; while thanks to recent development performing such experiment on conventional X-ray source seems feasible in relatively short future.

Indeed, the technique does not need any sample preparation or chemical injection and the deposited radiation dose could drastically be reduced with iterative reconstruction algorithm Experimental set up of the X-ray Phase Contrast Image A wiggler source produced a broadspectrum synchrotron ration which was filtered to a narrow energy band at 52 keV by a double bent Silicium crystal. Sample was 11 meters from the detector in order to broaden x-ray interference. A gadolinium oxysulfide phosphor screen was used as scintillator to convert the x-ray to visible light. Images in visible light were then recorded indirectly by a sCMOS camera coupled with an optical system giving a voxel size of 6.1×6.1×6.1μm 3 .

Morphological Metrics

3 , 7 . 8 .

 378 Imaging modalities are becoming a gold standard to quantify the effectiveness of such therapies. Thus, it is crucial to develop, robust and reproducible joint imaging techniques with a high spatial resolution and sensitive to soft and hard tissues. Current clinical and preclinical imaging modalities have limitations in the detection of early cartilage and bony changes. Indeed, conventional X-ray absorption based Computed Tomography (CT) allows clear visualization of bone tissues but provides reduced sensitivity to soft tissues Cartilage is indeed poorly visible in conventional micro-CT and it mainly shows advanced and irreversible cartilage degradation resulting in joint space narrowing. Changes in the composition of joint cartilage or soft tissues are usually rather evaluated using Magnetic Resonance Imaging (MRI) using sequences such as ultrashort-TE 9 . Yet, images obtained by MRI struggle to render properly bony changes and microcalcifications 10

13 .

 13 These methods, called Phase Contrast Imaging (PCI) in comparison to the conventional method based on absorption. PCI has a strong correlation with MRI in measuring cartilage thickness and with conventional CT in detecting subchondral bony changes ,14,15 . It is indeed possible to visualize simultaneously and in details the high absorbing tissues like bone, and the low-absorbing tissues like cartilage and soft tissue (synovial membranes, muscles ...) as demonstrated by

Horng

16

 16

3 ,

 3 pulse sequence was acquired (echo time = 0.00813 ms; repetition time = 4.0 ms; flip angle = 5 • ; acquisition matrix = 300 × 300 × 300, average = 4). Such acquisition allows acquiring a volume of 15×15×15mm

3 )

 3 gives a poor geometric representation of the overall bone, meniscus and tibial ligaments and underestimate the total number of calcifications (Ncalcification MRI = 57, Ncalcification CT = 314 vs Ncalcification PhaseContrast = 329). Due to poor contrast within the bone structure, calcified cartilage, cortical and trabecular bone appear homogeneous with the MRI technique.

  computed three-dimensional based measurements on the PCI images that are not accessible with histology. We first computed the surface roughness (estimated as the local curvature of a surface) of different interfaces: the calcified cartilage/articular cartilage interface and the calcified cartilage/ subchondral bone interface. The Figure 4A-D shows such measurements where the roughness is projected onto the surfaces (represented in false colors) of both the calcified cartilage (A and C) and subchondral bone (B and D) in an example of control (A and B) and MIA (C and D) joints. The subfigure 4E-H, together with an exemplary image, shows the roughness distributions restricted to the condyles positions. Figure 4E and F is the measurement performed on the femur, and G and H on the tibia. Note that only the femur subchondral bone displays a significant increase in curvature variability on the MIA-OA model (curvatureσ Curv OA

Figure 3 ±

 3 Figure 5E and G, the roughness on the tidemark between the calcified cartilage and subchondral bone is significantly different in between the two measured groups with a larger variability in curvature surface for the MIA model for both and tibia. The calcification distribution is significantly different between the model and the control (nb MIA = 54.8 ± 21.5 vs nb Ctr = 12.1 ± 4.21) (Figure 5H). The volumes distribution seems bimodal in the MIA model with a total of V Ctr = 2.77 × 10 3 ± 4.26 × 10 2 μL vs V MIA = 3.70 × 10 4 ± 6.84 × 10 3 μL. As expected, the smallest calcifications are mainly distributed in the tightest spaces in between the two condyles. The total volume of calcified cartilage was also higher in the MIA group compare to the control with a mean volume per animal of V MIA = 11.0 ± 2.45 × 10 -2 mm 3 vs V Ctr = 5.84 ± 0.86 × 10 -2 mm 3

20 . 21 . 20 .

 202120 Although not fully depicting the disease progression in human, this MIA-OA model induces chondrocytes cell death within a week, a rapidly evolving OA and formation of osteophytes in rodent The chemical agents (Mono-Iodo-Acetate (MIA), papain, collagenase) induce very serious lesions with complete and rapid destruction of the cartilage making it impossible to study the early phases of OA disease. Despite that, to depict the joints tissue elements with enough contrast at the micro scale in such a small model remain highly challenging with conventional pre-clinical imaging modalities. In this study the histological lesions are severe and confirm that an OA has been induced. Some samples are close to the maximum of the OARSI scoring system with large depletion of proteoglycan staining, while other have a lack of articular cartilage. Mice calcified cartilage is proportionally larger in mice than in human 17 and mice MIA-OA model is mildly affected by mechanical stress due to the animal weight We nevertheless found thickening of the calcified cartilage and a rougher surface of the tidemark in the OA-model. As expected on an MIA-OA model 22

32 .

 32 feasibility of performing phase contrast with clinical sources. Unfortunately, in these studies the application of PCI remains in 2D due to the additional optics used. A simple approach using a random modulator[START_REF] Paganin | Single-image geometric-flow x-ray speckle tracking[END_REF][START_REF] Labriet | 3d histopathology speckle phase contrast imaging: from synchrotron to conventional sources[END_REF][START_REF] Zdora | X-ray phase-contrast imaging and metrology through unified modulated pattern analysis[END_REF] has recently shown that PCI can be transferred on conventional source with short acquisition time and reduced radiation dose to the sample To conclude, PCI provides in a single examination, a better and a more detailed depiction of the different tissues of small animal models. The sensitivity of PCI makes possible a diagnosis and staging tool of the joint deterioration to support the development of new pre-osteoarthritic treatments and could in the future be applied to clinical studies.

Figure 1 .

 1 Figure 1. Representative images of one osteoarthritis knee sample observed by each imaging technique:A:MRI with a ultra-short echo time sequence (echo time = 0.00813 ms; repetition time = 4.0 ms; flip angle = 5 • ; voxel size=50 × 50 × 50 μ m 3 ), B: Conventional X-ray micro-tomography Image (Source voltage = 60 kVp; voxel size =6.06 × 6.06 × 6.06 μ m 3 ) and C: Synchrotron X-ray phase contrast imaging (source energy = 52 keV; voxel size = 6.1 × 6.1 × 6.1 μ m 3 ), D,E: Phase contrast Image magnification with a soft ( D ) and hard ( E ) tissue contrast window.

Figure 2 .Figure 3 .

 23 Figure 2. Representative images acquired by PCI (A-D) and their corresponding histological slices (E-J) of the control (A,C,E,G,H) and the MIA-OA model (B,D,F,I,J). Magnified images of the lateral condyle (yellow and black square) highlight the subchondral bone ( †), calcified cartilage (*), uncalcified cartilage (red dotted line) and example of chondrocytes (γ)

Figure 4 .

 4 Figure 4. Quantification of knees structures roughness and calcifications. A-H: Computed roughness of the surface linemark between the calcified cartilage and cartilage (A and C) and Subcondral bone and calcified cartilage (B and D). A and D: 3D mapping of the calculated roughness E-H: Roughness Distribution calculated in between the two condyles in the Femur (E and F) and Tibia (G and H) I-J: Quantification of all the labeled calcifications . I and J: 3D rendering of the detected calcification, on the control knee (I) and MIA (J). K-L: 2D representation of all the condrocite coronaly projected with surface area the volume of each condrocites.

Figure 5 .

 5 Figure 5. Quantification of knees structures morphology. Comparison made on the MIA model (n=6) and the controlateral legs (n=6). A : Computed mean and standard deviation inter-space between the condyles contact points and tibia (*: p < 0.01). B and C Histogram of the thickness of the calcified cartilage for the femurs and the tibias. D, E, F, G: Computed surface roughness on the calcified/uncalcified cartilage tidemark (D and F) and subchondral bone/calcified cartilage tidemark (E and G). H: Representation of the location and the number of calcifications (up) with their volume distribution (bottom).

Figure 6 .

 6 Figure 6. Experimental set up of the X-ray Phase Contrast Image A wiggler source produced a broadspectrum synchrotron ration which was filtered to a narrow energy band at 52 keV by a double bent Silicium crystal. Sample was 11 meters from the detector in order to broaden x-ray interference. A gadolinium oxysulfide phosphor screen was used as scintillator to convert the x-ray to visible light. Images in visible light were then recorded indirectly by a sCMOS camera coupled with an optical system giving a voxel size of 6.1×6.1×6.1μm 3 .

Table 1 .

 1 C Femur [mm 2 ] Calci f ied Cartilage C Tibia [mm 2 ] Calci f ied Cartilage C Femur [mm 2 ] Subcondral BoneC Tibia [mm 2 ] Subcondral Bone All computed metrics on the knees database (control n= 6 and MIA n = 6). With D: Interspace the tibia and the femur; T : local thickness of the calcified cartilage; V : volume; C: Curvature of the specified surface; N: Number of calcification.Each * a statistical difference in between the control and MIA group with p < 0.001

		Control (n=6)	MIA (n=6)	p<0.01
	D BothCondyles [μm]	56.3 ± 6.6	37.4 ± 13.9
	D MedialCondyle [μm]	55.6 ± 9.1	25.6 ± 6.8	* * *
	D LateralCondyle [μm]	56.7 ± 4.1	70.7 ± 4.3
	T Femur [μ m]	24.2 ± 7.3	47,7±15.4
				* *
	T Tibia [μm]	25.1 ± 8.1	44.4 ± 17.2
	V CacifiedCartilage [μm]	5.84 ± 0.86	11.0 ± 2.45	*
		4.32 ± 1.09 × 10 -2	4.57 ± 1.72 × 10 -2
		4.75 ± 1.14 × 10 -2	4.81 ± 1.82 × 10 -2
				* *
		6.78 ± 2.25 × 10 -2	12.7 ± 3.54 × 10 -2
		7.54 ± 2.47 × 10 -2	11.6 ± 2.85 × 10 -2
	N Calcification	12.1 ± 4.21	54.8 ± 21.5	*
	V Calcification [μL]	2.77 ± 0.462 × 10 3 3.70 ± 0.684 × 10 4 *

Acknowledgements

The authors acknowledge the ESRF for granting beamtime and their support during the experiment. Part of this study was funded by the Idex Grenoble grant IRS Valium. The authors acknowledge C. Ferrero for scientific discussion, L. Salvo P. Lhuissier for beamtime and help on the conventional microCT device.

Author contributions

Study conception and design: LB, BF, HRL, SD, BLa, BLe, EB; Data acquisition: LB, BF, HRL, EB, BLe; Data analysis and interpretation: LB, BLe, EB; Drafting the article: LB, BF, HRL, SD, BLa, BLe, EB; Article final approval: LB, BF, HRL, SD, BLa,BLe, EB.

Conflict of Interest.

The authors declare no conflict of interests


Emeline Seurat

Anthony Verdin

Fabrice Cazier

Dominique Courcot

Richard Fitoussi

Katell Vié

Valérie Desauziers

Isabelle Momas

Nathalie Seta

Sophie Achard
email: sophie.achard@u-paris.fr

Influence of the environmental relative humidity on the inflammatory response of skin model after exposure to various environmental pollutants

Keywords: 3D-Skin model Air Liquid exposure Environmental pollutants Environmental humidity Inflammatory response DMSO: dimethyl sulfoxide, IL: Interleukin, MMPs: matrix metalloproteinases, MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylte-trazolium bromide, PM: particulate matter, RH: relative humidity, RHE: Reconstructed Human Epidermis, ROS: reactive oxygen species, SDS: sodium dodecyl sulfate, TS: tobacco smoke, VOCs: volatile organic compounds

The skin is an essential barrier, protecting the body against the environment and its numerous pollutants. Several environmental pollutants are known to affect the skin, inducing premature aging through mechanisms including oxidative stress, inflammation, and impairment of skin functions. Even climate conditions can impact the skin. Therefore, using a Reconstructed Human Epidermis (RHE), we tested the effect of two samples of fine particulate matters (PM0.3-2.5 -one metals-rich sample and the other organic compounds-rich), two Volatile Organic Compounds mixtures (VOCs -from a solvent-based paint and a water-based paint) and Tobacco Smoke (TS). All pollutants affected cellular functionality, but to a lesser extent for the water-based paint VOC. This effect was enhanced when RHE were preconditioned for two hours by a semi-dry airflow (45% relative humidity) before pollutants application, compared to preconditioning by a humid airflow (90% relative humidity). In the absence of preconditioning, IL-1α, IL-6, IL-8, and RANTES were almost systematically induced by pollutants. When RHE were preconditioned by a semi-dry or humid airflow before being subjected to pollutants, the increase of IL-1α, IL-8, and RANTES falls into two groups. Similarly to RHE not treated with pollutants, RHE treated with VOCs after preconditioning by a semi-dry airflow showed increased IL-1α, IL-8, and RANTES release. On the contrary, RHE treated with PM or TS after preconditioning by a semidry airflow show a lower increase in IL-1α, IL-8, and RANTES compared to preconditioning by a humid airflow. The effect of real environmental relative humidity conditions of the air, combined with acute exposure to various environmental pollutants, seemed to relate mainly to structural changes of the skin, determining the outcome of E. Seurat et al.

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the inflammatory response depending on the physicochemical characteristics of 47 pollutants.

Introduction

Air pollution -the degradation of air quality due to harmful gases, chemicals, biological contaminants, and/or particles -is associated with a variety of acute and chronic illnesses leading to increased morbidity and mortality [START_REF] Hoek | Long-term air pollution exposure and cardio-respiratory mortality: a review[END_REF][START_REF] Willers | Fine and coarse particulate air pollution in relation to respiratory health in Sweden[END_REF]. Like the airway epithelium, the skin is constantly exposed to the environment and its pollutants. It is essential in protecting the body and many factors can affect it.

An obvious intrinsic factor that affects the skin is aging. It is visually characterized by the appearance of wrinkles, thinner epidermis, cutaneous dryness, ptosis, loss of elasticity, dark spots, and uneven pigmentation. Central to the process are the increase of oxidative stress, the increase in the activity of matrix metalloproteinases (MMPs) leading to degradation of the extracellular matrix, and the increase in the expression of cytokines and interleukins [START_REF] Haydont | Age-related evolutions of the dermis: Clinical signs, fibroblast and extracellular matrix dynamics[END_REF][START_REF] Parrado | Environmental Stressors on Skin Aging. Mechanistic Insights[END_REF]. If intrinsic factors can accelerate these processes, extrinsic factors are also important [START_REF] Makrantonaki | Skin aging: Molecular understanding of extrinsic and intrinsic processes[END_REF]. The effect of UVs is well known [START_REF] Jackson | Elderly and sun-affected skin. Distinguishing between changes caused by aging and changes caused by habitual exposure to sun[END_REF][START_REF] Flament | Effect of the sun on visible clinical signs of aging in Caucasian skin[END_REF]. Airborne pollutants also exert negative effects on the skin, especially on sensitive skin that can overreact to external aggression [START_REF] Krutmann | Pollution and skin: from epidemiological and mechanistic studies to clinical implications[END_REF].

For several years, works agreed that Tobacco Smoke (TS) has a strong impact on the skin. TS is a complex mixture of over 4,000 compounds (CO, HCN, benzene, formaldehyde, nicotine, phenol, polycyclic aromatic hydrocarbons, etc.) and only the particulate phase, which represents approximately 5% of the cigarette's total output, is visible as smoke (Hoffmann et al., 1997;[START_REF] Löfrth | Environmental Tobacco Smoke: Overview of Chemical Composition and Genotoxic Components[END_REF][START_REF] Auer | Heat-Not-Burn Tobacco Cigarettes: Smoke by Any Other Name[END_REF]. This complex mixture leads to premature skin aging, wrinkling, poor wound healing, squamous cell carcinoma, and chronic dermatoses [START_REF] Ortiz | Smoking and the skin[END_REF]. In vivo studies revealed E. Seurat et al. 6/39 that TS induces reactive oxygen species (ROS), affects collagen production, and increases the production of tropoelastin as well as MMPs, leading to the degradation of the extracellular matrix [START_REF] Morita | Tobacco smoke causes premature skin aging[END_REF]. We previously corroborate these results, using Reconstructed Human Epidermis (RHE), showing that, if two TS exposures are sufficient to alter the stratum corneum, a single one is enough to induce inflammatory cytokines, MMPs, and oxidative stress, as well as down-regulation of essential skin functions [START_REF] Lecas | In vitro model adapted to the study of skin ageing induced by air pollution[END_REF].

Outdoor air pollution is made up of gases (O3, CO2, CO, SO2, and NOx) and particulate matters (PM). It is considered the world's largest environmental health risk factor due to fine (PM2.5) and ultrafine (PM0.1) particles (WHO, 2018;[START_REF] Loomis | The International Agency for Research on Cancer (IARC) evaluation of the carcinogenicity of outdoor air pollution: focus on China[END_REF]. In addition to the respiratory diseases and premature death they induce (Beelen et al., 2014;[START_REF] Raaschou-Nielsen | Air pollution and lung cancer incidence in 17 European cohorts: prospective analyses from the European Study of Cohorts for Air Pollution Effects (ESCAPE)[END_REF], PM are responsible for premature skin aging, appearance of pigmentation spots, decrease in squalene (a major lipidic component of sebum) and vitamin E, as well as of the increase of lactic acid and melanin, these later indicating oxidative stress [START_REF] Schikowski | Air Pollution and Skin Aging[END_REF]. Using RHE we showed that exposure to PM0.3-2.5 affects cellular functionality, induces the release of inflammatory cytokines, and leads to oxidative stress [START_REF] Verdin | An in vitro model to evaluate the impact of environmental fine particles (PM0.3-2.5) on skin damage[END_REF]. PM also have a negative impact on fundamental skin functions: anchorage, cell differentiation, cornification/skin desquamation, and apoptosis.

Another source of pollution under scrutiny are Volatile Organic Compounds (VOCs). Defined as organic compounds with a high vapor pressure at normal room temperature (EU, 2010), they represent a numerous, varied, and ubiquitous group of substances.

Because of the time people spend inside, VOCs emanating from indoor air pollution are of particular concern. Their sources are numerous including building material, E. Seurat et al.

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equipment, cleaning products, or even combustion processes such as cooking [START_REF] Kostiainen | Volatile organic compounds in the indoor air of normal and sick houses[END_REF]WHO, 2010). Some of them (toluene, benzene, ethylbenzene, and xylene for instance) are known to have serious adverse effects on human health. So far, the effects of VOCs on the skin is poorly studied. Still, using keratinocytes, [START_REF] Dezest | Oxidative damage and impairment of protein quality control systems in keratinocytes exposed to a volatile organic compounds cocktail[END_REF] suggested that VOCs would play a role in premature skin aging. Indeed, they show that VOCs inhibit the proteasome, inducing apoptosis, DNA damage, and protein oxidation.

Oxidative stress, inflammation, and metabolic impairments seem to be common mechanisms of skin aging and pollution-induced premature skin aging. Even climate conditions -relative humidity, temperatures -impact skin properties and barrier function and may modify skin reactions [START_REF] Singh | Climate and skin function: an overview[END_REF][START_REF] Cau | Lowering relative humidity level increases epidermal protein deimination and drives human filaggrin breakdown[END_REF]. Therefore, we evaluated the effect of several pollutants using an RHE system, focusing on inflammation markers. The pollutants tested are two PM0.3-2.5 samples with different characteristics, VOCs samples from two different paints and TS. We especially focused on how semi-dry/humid conditions can affect the inflammation response induced by pollutants.

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Materials and Methods

In vitro skin model

The in vitro skin model used is a reconstructed human epidermis system (RHE) from SkinEthic™ (Lyon, France; [START_REF] Rosdy | Terminal epidermal differentiation of human keratinocytes grown in chemically defined medium on inert filter substrates at the airliquid interface[END_REF][START_REF] Rosdy | Production of basement membrane components by a reconstructed epidermis cultured in the absence of serum and dermal E. Seurat et al. 35/39 factors[END_REF]. It provides a differentiated 3D epidermal tissue from human keratinocytes for which signed informed consent and ethical approval were obtained by the supplier. All experiments were conducted on a single batch (MF22-5).

Culture media, growth medium for non-experiment days, and maintenance medium for experiment days were supplemented with 1% of Penicillin-Streptomycin mixture (penicillin: 100 μg/mL; streptomycin: 100UI/mL). Upon reception, RHEs were placed in a 24-well plate with growth medium on the basal side and maintained in an incubator at 37 °C, 5% CO2, and 95% relative humidity. Experiments were carried out 24h after RHE-acclimation.

Fine particulate matters (PM0.3-2.5)

Two samples of fine PM, having the same aerodynamic diameter (0.3 to 2.5µm, PM0.3-2.5) and similar granulometric profile, but differing in their sampling origin and their chemical composition were tested: PM from Cotonou (Benin, West Africa) that are rich in metals and inorganic elements (PMm -metal-rich PM), and PM from Dunkerque (France) that are rich in organic compounds (PMo -organic-rich PM). The collection and characterization of both PM were already published [START_REF] Cachon | Proinflammatory effects and oxidative stress within human bronchial epithelial cells exposed to atmospheric particulate matter (PM2.5 and PM>2.5) collected from Cotonou, Benin[END_REF][START_REF] Dergham | Temporal-spatial variations of the physicochemical characteristics of air pollution Particulate Matter (PM2.5-0.3) and toxicological effects in human bronchial epithelial cells (BEAS-2B)[END_REF]. A summary of their composition is presented in Table 1 and detailed analysis in Table S1.

Aliquoted and kept at -20°C after collection, PM were thawed and suspended in the maintenance culture medium containing 1% of Penicillin-Streptomycin and 1% E. Seurat et al. 9/39 fungizone (Thermo Fisher Scientific, Waltham, MA, USA). Twenty-four hours before experiments, PM suspension was sonicated (50W, 10 min, Bath sonicator Branson CPXH, Dutscher, France) and 30 µL were deposited onto the apical side of RHEs.

Three concentrations -3, 15, and 30 μg/cm 2 -as previously described [START_REF] Verdin | An in vitro model to evaluate the impact of environmental fine particles (PM0.3-2.5) on skin damage[END_REF] were tested. Medium without PM was used as a negative control.

Volatile organic compounds (VOCs)

VOC atmospheres were generated from two different paints, which containers have never been opened). VOCwb (water-based) are from an aqueous paint (Brillant Intérieur Acrylique, Auchan, France) bought in 2014, after the French regulation governing The protocol applied to generate VOCs atmospheres was inspired by the emission standard test (ISO 16000-9, ISO, 2006a) and the paints were prepared according to ISO 16000-11 (ISO, 2006b). Briefly, paint was spread onto a 150 cm² glass plate that was then placed in an 11 L sealed generation chamber under a steady airflow (20-25°C, 79 ± 8% humidity and a rate of 0.5 L/h [START_REF] Ricquebourg | Airway epithelium co-cultured with immune cells for a better assessment of the low dose effects of environmental pollutants on the inflammatory response[END_REF]. The VOCs atmosphere was stabilized for 3 days before exposure. Samplings of VOCs from paint were chemically controlled by vacuum vial SPME sampling [START_REF] Desauziers | SPME-adapter: a rapid sampling for the analysis of VOCs traces in air[END_REF] followed by GC/MS/FID analysis. The concentrations of VOCwb and VOCsb E. Seurat et al.

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were determined as toluene equivalent from the FID signal [START_REF] Badji | Under glass weathering of hemp fibers reinforced polypropylene biocomposites: Impact of Volatile Organic Compounds emissions on indoor air quality[END_REF] and are summarized in Table 2 and detailed in Table S2.

On the day of exposure, the generation chamber was connected to the Vitrocell ® dynamic exposure system (Vitrocell System ® GmbH, Waldkirch, Germany). This system contains six exposure chambers, each one with an insert with the RHE tissue and a trumpet. For one hour, a 2mL/min airflow was created to deliver the VOC atmosphere through the trumpet, onto the apical side of the RHE that was maintained at 37°C thanks to a water bath [START_REF] Persoz | An in vitro model to evaluate the inflammatory response after gaseous formaldehyde exposure of lung epithelial cells[END_REF]. An atmosphere without VOCs, delivered with the same dynamic system, was used as a negative control.

Tobacco smoke (TS)

TS that is a complex mixture containing polycyclic aromatic hydrocarbons, volatile organic compounds, and PM present in air pollution was used as positive environmental pollution control. For this purpose, mainstream tobacco smoke (TS) was generated by the combustion of a French brand cigarette composed of 87% tobacco, 6.5% flavors agents, and 6.5% cigarette paper. It produces 7 mg of tar, 0.6 mg of nicotine, 9 mg of carbon monoxide and comprises two parts, gas and particulate matter. Using a static system adapted from the Vitrocell Cloud™ chamber (Vitrocell System ® GmbH, Germany), fresh TS was delivered to the apical face of RHE at a rate of 25 mL per puff with a total of 10 puffs [START_REF] Lecas | In vitro model adapted to the study of skin ageing induced by air pollution[END_REF][START_REF] Bardet | Human Reconstituted Nasal Epithelium, a promising in vitro model to assess impacts of environmental complex mixtures[END_REF]. The dose delivered corresponds to one cigarette, with an average of 200 μg/cm 2 of particulate matters. Similarly to RHEs treated with VOCs, the air atmosphere used to generate TS was used as a negative control.

Exposure of RHEs to pollutants

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To compare the effect of the sole pollutants, RHEs were exposed to pollutants as described above, then incubated for 24h in classical culture conditions (incubator at 37°C, with 5% CO2, and 90% relative humidity). RHEs not subjected to pollutant were used as negative controls (Fig. 1A,1C). For all experimental series, at the end of the incubation period, culture media were collected to assess the immune response by quantifying cytokines/chemokines release, and RHEs were harvested to perform MTT assay.

Preconditioning of RHE to semi-dry/humid airflow followed by exposure to pollutants

To assess the role of humidity on the impact of pollutants, RHE were preconditioned by a semi-dry or humid airflow for 2h before being incubated with pollutants. RHE maintained at 37°C thanks to a water bath, were subjected to different airflows. The airflow was delivered using the Vitrocell ® Humidification Station (Vitrocell Systems ® GmbH, Waldkirch, Germany) that was supplying semi-dry air (24°C, 5% CO2 and 45% relative humidity (RH) similarly to European conditions like in Dunkerque city) or humid air (24°C, 5% CO2 and 90% RH representative of conditions in South America, Asia or Africa, like in Cotonou city). Then, RHEs were exposed to pollutants as described above, and further incubated for 24 h in classical culture conditions (37°C, with 5% CO2, and 95% RH) before performing MTT assays on RHE and quantifying cytokines/chemokines released in the culture media.

The protocol of exposure is described in Fig. 1B. Various controls were considered and are summarized in Fig. 1C. Negative controls: RHEs preconditioned by the humid or semi-dry airflow but not exposed to pollutants, and RH controls: RHEs exposed to the E. Seurat et al.

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Cellular functionality: MTT assay

Cellular functionality was assayed on the basal side of RHE relying on the ability of the mitochondrial succinate dehydrogenase to convert MTT 342,5-diphenylte-trazolium bromide) into a formazan product that has a maximum photometric absorbance at 490 nm [START_REF] Persoz | An in vitro model to evaluate the inflammatory response after gaseous formaldehyde exposure of lung epithelial cells[END_REF]. Briefly, 24h after exposure, the culture medium was removed, 500 µL of MTT was added to the bottom of the well containing the RHE. After two hours of incubation (37°C, with 5% CO2, and 95% relative humidity), DMSO (500µL) was added to the bottom of the well, reagents were homogenized by shaking and transferred to 96-well plates. Absorbance was read using a microtiter plate reader (Multiskan ® EX, Thermo Scientific, Waltham, MA, USA).

Mapping of cytokine production: Q-Plex TM analysis

Q-Plex TM quantitative ELISA-based chemiluminescent assay (Quansys, Logan, UT, USA) were performed according to the manufacturer's instructions, using the profiling service of Tebu-bio (Le Perray-en-Yvelines, France). Nine cytokines (Q-Plex Human Cytokine-Inflammation #110433HU -IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IFNy and TNFα) and eight chemokines (Q-Plex Human Chemokine Inflammation #110451HU -Eotaxin, GROα, I-309, IP-10, MCP-1, MCP-2, RANTES and TARC) were measured. The analysis was performed by quantifying the intensity of the chemiluminescent signals on the Tebu-bio technological platform using both Q-View™ and Quansys Software. Sensitivity ranges between 1 pg/mL and 30 pg/mL depending on the considered analyte.

Specific cytokine/chemokine production: ELISA analysis

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Two cytokines and one chemokine were assessed using Human DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) interleukin 1α (IL-1α/IL-1F1, #DY200), interleukin 8 (IL-8/CxCL8, #DY208) and RANTES (CCL5/RANTES, #DY278). Absorbance was determined using an ELISA-reader (Multiskan ® EX, Thermo Scientific, Waltham, MA, USA) at 450 nm and 540 nm to correct the optical imperfections in the plate.

Calibration curves were calculated using Ascent Software ® (Thermo Fisher Scientific, Waltham, MA, USA). Concentrations are expressed in pg/mL and the lower limits of quantification are 31.26, 7.81, 156 pg/mL for IL-8, IL-1α, and RANTES, respectively.

Statistical analysis

All results are reported as mean ± standard deviation. Except for Q-Plex TM analysis that was performed only once with duplicated quantification, all other experiments were performed at least in two independent experiments, each with duplicated quantification.

The exact number of independent experiments (n) is indicated in the legend of the figures. After verifying the Normal distribution of results using the Shapiro-Wilk test (p<0.05), statistical differences were analyzed using one-way ANOVA followed by Tukey HSD (Honestly Significant Difference) test. p<0.05 was considered significant. E. Seurat et al.

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Results

RHE functionality upon exposure to pollutants

RHEs were exposed to various environmental pollutants: two PM0.3-2.5 samples -PMm, rich in metals and inorganic elements, and PMo, rich in organic compounds -two VOC mixtures -VOCwb, emanating from a water-based paint, and VOCsb, from a solventbased paint -and TS.

Results of the MTT assay (Fig. 2) reveals that the pollutant which most affects RHE is TS, inducing a significant -43.9% (p<0.0001) decrease in cell functionality compared to the negative control. Moreover, both PM were found to alter the functionality of RHEs.

The effect is dose-dependent and statistically significant from the lowest exposure doses to 3 µg/cm 2 of PMm (-10.5%, p=0.0155) or 15 µg/cm 2 of PMo (-15.4%, p<0.0001). The negative effect is higher when RHEs are exposed to PMm than to PMo, reaching -39.4% (p<0.0001) for exposure to 30 µg/cm 2 of PMm, while the decrease induced by PMo is of -26.7% (p<0.0001). Among the two VOCs tested, only VOCsb affects RHEs, leading to -8.7% (p=0.0213) in cell functionality, the -4.4% decrease induced by VOCwb being not significant (p=0.4543).

RHE functionality upon preconditioning in a semi-dry or humid airflow followed by exposure to pollutants

To evaluate the impact of airflow with different relative humidity on the effect of pollutants, we preconditioned RHEs by submitting them to a semi-dry (45% relative humidity -RH) or a humid (90% RH) airflow for 2 hours prior exposure to pollutants. Controls included negative controls, preconditioned by the semi-dry or humid airflow but not subjected to pollutants, and RH controls, incubated with pollutants but not to E. Seurat et al.

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preconditioning by the humid/semi-dry airflow (kept in the incubator at 37°C with 90% RH).

Results (Fig. 3) showed that, for the negative control, preconditioning by the humid airflow does not affect RHE compared to the RH control. This lack of effect of preconditioning by the humid airflow is also observed when comparing RHE incubated with pollutants with their respective RH controls. Only preconditioning by the semi-dry airflow affects the results of the MTT test for both, the negative control and the RHE treated with the various pollutants. The decrease is always significant when results are compared to that of RHE preconditioned by the humid airflow and almost always when compared to the respective RH controls (the exception being PMm). Still, within series, the decrease in cellular functionality induced by the semi-dry preconditioning compared to preconditioning by the humid airflow remains generally mild (negative control: -12.9%, p=0.0443; PMm: -16.7%, p=0.0242; PMo: -19.8%, p=0.0014; VOCwb: -23.9%, p<0.0001; VOCsb: -26.6%, p<0.0001), except for TS for which it reaches -34.1% (p=0.0007).

Inflammatory response upon exposure to pollutants

We have then investigated the inflammatory response induced by pollutants by monitoring the production of nine cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IFNα, TNFα) and eight chemokines (Eotaxin, GROα, I-309, IP-10, MCP-1, MCP-2, RANTES, TARC). The results are presented in Fig. 4. IL-1α, IL-6, IL-8, and RANTES are the only cytokines/chemokines almost systematically and significantly induced by a factor of 1.3 to 2.7 by pollutants: PM at 15 µg/cm 2 and over, VOCs and TS. Even if VOCwb does not significantly induce IL-1α/IL-6 and TS does not significantly induce IL-8, the probabilities are at the limit of E. Seurat et al.

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significance for IL-1α and VOCwb (p=0.0535), as well as for IL-8 and TS (p=0.0903).

For both PM a dose-dependent effect is clearly observed for IL-1α, IL-6, IL-8, and RANTES. Besides, the induction of the three cytokines/chemokines seems to be higher for PMm (x2.2 folds for IL-1α, x2.7 folds for IL-6, x2.7 folds for IL-8 and 2.1 folds for RANTES with 30 µg/cm 2 ) than for PMo (x2.1 folds for IL-1α, x1.9 folds for IL-6, x2.1 folds for IL-8 and 1.8 folds for RANTES with 30 µg/cm 2 ).

In these experimental series, none of the pollutants induced IL-1β, IL-4, Eotaxin, I-309, MCP-2 or TARC, and the induction of IL-2, IL-10, IFNα, TNFα, GROα, IP-10, MCP-1 depends on the pollutant. Still, overall PMm is the pollutant inducing the most cytokines and chemokines (10 among the 17 tested) while VOCwb is the one inducing the least (5), VOCsb, and TS being intermediate.

Inflammatory response upon preconditioning in a semi-dry or humid airflow followed by exposure to pollutants

To analyze the effect of airflows with different relative humidity on the inflammatory response induced by pollutants, we preconditioned RHEs for two hours using a semidry or humid airflow before subjecting them to pollutants. For this analysis, we focused on IL-1α, IL-8, and RANTES that were induced by most pollutants tested. Similarly to the study of the cellular functionality of both, semi-dry/humid airflow and pollutants, the controls encompassed negative controls, subjected to preconditioning by the different airflows but not to pollutants, and RH controls, subjected to pollutants but not to preconditioning by the airflows. The results are presented in Fig. 5.

In the negative controls, comparison of the amount of cytokines/chemokine released by the RH controls and by RHEs preconditioned by the 90% RH airflow shows that preconditioning by the humid airflow significantly increases the release of IL-1α and E. Seurat et al.

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IL-8 by a factor of 1.9, while, for RANTES, the increase is not significant. The increase in IL-1α and IL-8 is also observed when comparing RH controls treated with pollutants and RHEs which underwent both, preconditioning by the humid airflow and incubation with pollutants, but, in that case, the increase is also significant for RANTES. In addition, the fold change compared to RHE treated with pollutants but not preconditioned by the humid airflow is much higher, reaching over 10 folds for IL-8 induction by any of the PM and 3 to 5 folds for RANTES inductions by whatever VOCs.

For all negative controls, preconditioning in the semi-dry airflow significantly increases the release of IL-1α, IL-8 and RANTES compared to the RH controls. Except for IL-1α, this increase is significantly higher than that obtained upon preconditioning by the humid airflow. When RHEs are treated with both, preconditioning by the semi-dry airflow and either PM, the release of IL-1α, IL-8 and RANTES is higher than in the RH controls, but overall significantly lower than when RHEs are preconditioned by the humid airflow. For TS, combination with preconditioning by the semi-dry airflow acts similarly to PM on IL-1α, IL-8, and RANTES. Contrary to PM and TS, preconditioning by the semi-dry airflow and either VOC, VOCwb, or VOCsb, leads to an increase in IL-1α, IL-8, and RANTES compared to preconditioning by the humid airflow. E. Seurat et al.

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Discussion

In recent years, several studies were devoted to the biological effect of air pollutants.

Many focused on small particulate matters (PM<2.5) due to their role in pulmonary morbidity and mortality. But PM are not the only source of air pollution and, with the increasing time spent inside, VOCs are also important to consider. Besides, the majority of the studies focuses on the pulmonary epithelium. Little is known about the fate of the skin despite the fact it is the largest human organ and it is also constantly exposed to the environment. Therefore, we wanted to better understand the impact of air pollutants on the skin and, especially, to investigate how an environmental factor such as humidity affects the response to pollutants knowing that changes in relative humidity are sufficient to impact the skin [START_REF] Singh | Climate and skin function: an overview[END_REF][START_REF] Cau | Lowering relative humidity level increases epidermal protein deimination and drives human filaggrin breakdown[END_REF].

Among the pollutants we tested, TS was primarily used as a positive control. This pollutant is a complex mixture containing over 4,000 gaseous and particulate compounds, including VOCs and PM. Its effect on premature skin aging is already well documented and we previously showed, using the very same RHE system, that it induces morphological alterations, oxidative stress, leading to the production of inflammatory cytokines [START_REF] Lecas | In vitro model adapted to the study of skin ageing induced by air pollution[END_REF]. Therefore, our primary interest was the response induced by two types of pollutants, fine particles (PM0.3-2.5) and VOCs, with two different samples for each. Both PM samples present similar granulometric profiles, but show very different chemical compositions, therefore enabling us to evaluate the impact of chemicals adsorbed to the particles. PMm are originating from a highly polluted urban region of Sub-Saharan Africa (Cotonou, Benin). They are characterized by their extreme richness in metallic elements as the region where they were collected is surrounded by soils with high concentrations of iron oxide, aluminum oxide, and heavy E. Seurat et al.

19/39 metals [START_REF] Cachon | Proinflammatory effects and oxidative stress within human bronchial epithelial cells exposed to atmospheric particulate matter (PM2.5 and PM>2.5) collected from Cotonou, Benin[END_REF]. On the contrary, PMo are from a region of France (Dunkerque) with intense industrial activities and heavy vehicle traffic, which their richness in organic compounds reflects [START_REF] Dergham | Temporal-spatial variations of the physicochemical characteristics of air pollution Particulate Matter (PM2.5-0.3) and toxicological effects in human bronchial epithelial cells (BEAS-2B)[END_REF]. The last two pollutants we tested are VOCs. Much less studied, we focused on VOCs from a solvent-based paint that was manufactured before the French regulation limiting VOCs in construction and decoration products and that liberates large amounts of various VOCs. The second source of VOCs is a recent water-based paint manufactured after the French regulation and showing reduced VOCs emissions.

All these pollutants are real environmental pollutants. In addition, the exposure system we set up is realistic in the sense that the pollutants are delivered to the apical side of RHEs, therefore mimicking a real-life situation. Still, it is inhomogeneous in its exposition due to the physical nature of the different pollutants tested. As a matter of fact, PM are applied topically and remain on RHE for 24 hours before analysis, making it a static system. On the other hand, VOCs are delivered dynamically, via a one-hour airflow followed by a 24-hours incubation before analysis. The system we used for TS is intermediate. Fresh TS is generated in a chamber where its compounds are allowed to interact with, and sediment onto RHEs. Therefore, it is more of a static system that favors the action of the PM constituents that will stay on RHEs, rather than that of its gaseous phase for which the contact is time-limited. Another source of inhomogeneity is the dose of pollutants tested. If the doses of PM and TS we tested are much higher than normal exposures, as frequently in in vitro studies, those of VOCs are, on the contrary, very low: 6.1 µg/liter for VOCwb and 183.5 µg/liter for VOCsb, therefore 0.73 µg and 22.0 µg respectively during the one-hour exposure. These low doses are due to the fact that we followed the ISO standards to generate the VOCs samples. E. Seurat et al.

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Not only does our experimental setup show heterogeneity in the testing of pollutants, but the pollutants we tested are very different and interact differently with the skin. PM are a complex mixture of insoluble particles -the physical part -and soluble components absorbed to their surface -the chemical part. If PM can induce their effects through the release of soluble components that can enter the stratum corneum, the tiniest PM can enter the skin. Indeed, [START_REF] Jin | Urban particulate matter in air pollution penetrates into the barrier-disrupted skin and E. Seurat et al. 32/39 produces ROS-dependent cutaneous inflammatory response in vivo[END_REF] showed that PM can enter the skin by accumulating in hair follicles and sweat glands. In RHE, they were found to enter the upper layers in less than 24 hours (Magnani et al., 2015), which is not surprising regarding the fact that PM break the skin barrier by down-regulating filaggrin, thereby disrupting the tight skin junctions [START_REF] Pan | The impact of urban particulate pollution on skin barrier function and the subsequent drug absorption[END_REF][START_REF] Lee | Urban particulate matter down-regulates filaggrin via COX2 expression/ PGE2 production leading to skin barrier dysfunction[END_REF].

RHEs are deprived of hair follicles and sweat glands, but we incubated them with PM for 24 hours. Therefore, it seems likely that the toxicological endpoint we observe is due to both, the prolonged contact and/or diffusion of soluble components through the surface of the stratum corneum, as well as to the direct release of compounds absorbed to their surface within the upper layers of RHE. On their side, VOCs must have directly diffused into the upper layers of RHE. Indeed, the toxicity of hexane and toluene, two VOCs, relates to their lipophilicity and their accumulation in the lipid bilayer of cellular membranes [START_REF] Dreiem | Involvement of the extracellular signal regulated kinase pathway in hydrocarbon-induced reactive oxygen species formation in human neutrophil granulocytes[END_REF][START_REF] Pariselli | Effects of toluene and benzene air mixtures on human lung cells (A549)[END_REF]. Besides, VOCs are also known to affect the skin due to the ability of UV and NOx to transform them into photochemical oxidants, which seems unlikely to have played a major role considering our experimental setup.

Whatever the entry route of the pollutants, inhibition of the succinate dehydrogenase revealed by the MTT assay shows that PM, TS, and VOCsb affect RHEs. For the two PM, the alteration is dose-dependent, and the effect is stronger when RHE are treated E. Seurat et al.

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with PMm than with PMo. This may result from the high level of transition metals and other metals adsorbed on PMm (e.g. Al, Cu, Fe, Mn, Zn, Pb, etc.) that are powerful ROS inducers. Using the same exposure system, we previously showed that PMm are powerful inducers of oxidative stress [START_REF] Verdin | An in vitro model to evaluate the impact of environmental fine particles (PM0.3-2.5) on skin damage[END_REF]. The fact that polycyclic aromatic hydrocarbons absorbed to PM bind the aryl hydrocarbon receptor (AhR) to induce a signaling pathway leading to ROS formation and proinflammatory gene expression is now well established [START_REF] Esser | Council of 24 November 2010 on industrial emissions (integrated pollution prevention and control)[END_REF][START_REF] Parrado | Environmental Stressors on Skin Aging. Mechanistic Insights[END_REF]. Oxidative stress was also demonstrated for PMo using BEAS-2B bronchial epithelial cells [START_REF] Dergham | Temporal-spatial variations of the physicochemical characteristics of air pollution Particulate Matter (PM2.5-0.3) and toxicological effects in human bronchial epithelial cells (BEAS-2B)[END_REF]. Evidence of oxidative stress was also found for keratinocytes exposed to different VOCs [START_REF] Dezest | Oxidative damage and impairment of protein quality control systems in keratinocytes exposed to a volatile organic compounds cocktail[END_REF]. Oxidative stress and inflammation are tightly linked and, if ROS are not controlled, they lead to inflammatory cell and/or tissue injury (Lonkar and Dedon, 2010). In our case, all pollutants tested induce RANTES and almost all of them IL-1α as well as IL-8. Similarly to the cellular functionality, the effect is more pronounced for PMm, which also induces more cytokines and chemokines, than PMo. The induction of cytokines and chemokines production is also observed with the two VOCs and TS even if the range of cytokines and chemokines induced is more limited, especially for VOCwb.

When testing the influence of preconditioning by a humid versus a semi-dry airflow, results show that the semi-dry airflow impacts cellular functionality, whatever pollutant tested. Negative controls also revealed increased levels of IL-8 and RANTES upon preconditioning by the semi-dry airflow. If airflows differ between them by their relative humidity, they also differ from the negative control by the temperature (24°C).

Still, RHEs are maintained at 37°C and it is only their apical side that is subjected to this lower temperature. Hence, it sounds reasonable to assume that most of the effect we E. Seurat et al.

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observed is due to the differences in relative humidity, and that the increased levels of IL-8 and RANTES could be the first consequence of the initial increase in Transepidermal Water Loss (TEWL) followed by inflammatory cytokine stimulation as described in studies performed in dry conditions [START_REF] Singh | Climate and skin function: an overview[END_REF]. Still, results show that the short preconditioning period in semi-dry or humid air is sufficient to affect the reaction of RHE towards pollutants. Contrary to cellular functionality, the different preconditioning is also sufficient to reveal different effects depending on the type of pollutants. For both PM and TS, preconditioning by the semi-dry airflow tends to decrease the levels of IL-1α, IL-8, and RANTES compared to preconditioning by the humid airflow. The situation is just the opposite for the induction of IL-1α, IL-8, and RANTES by the two VOCs. Strikingly, both VOCs induce an almost identical response despite their very different qualitative and quantitative compositions. This response is similar to that of the negative control and suggests that, under the conditions used (low doses and short exposure), the effect of preconditioning by the semi-dry airflow primes over the response to VOCs. Taken together, these results suggest that the response of the skin to RH is the main driver of the toxicological endpoint and that its outcome depends mainly on the physical nature of the pollutant.

Several works studied the effect of dry conditions on the skin (reviewed in [START_REF] Engebretsen | The Effect of Environmental Humidity and Temperature on Skin Barrier Function and Dermatitis[END_REF][START_REF] Goad | Ambient Humidity and the Skin: The Impact of Air Humidity in Healthy and Diseased States[END_REF]. They show that human skin placed in dry conditions presents an increased stratum corneum thickness, an increased TEWL, a reduced elasticity, and an increased susceptibility to mechanical stress. In hairless mice, dry condition (3 days at 10% RH) induces reduced desmosomal degradation that was attributed to the reduced water content of the stratum corneum, leading to impaired desquamation and a scaly surface [START_REF] Sato | Dry condition affects desquamation of stratum corneum in vivo[END_REF]. Furthermore, the amount of IL-1α E. Seurat et al.

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was found higher in mice kept under dry conditions [START_REF] Ashida | Epidermal Interleukin-1α Generation Is Amplified at Low Humidity: Implications for the Pathogenesis of Inflammatory Dermatoses[END_REF]. RHE produced in semi-dry conditions (30-50% RH) also show increased TEWL, a thicker stratum corneum, and an increased level of epidermal protein deimination. These alterations lead to the breakdown of filaggrin, a filament-associated protein that binds to keratin fibers in epithelial cells [START_REF] Cau | Lowering relative humidity level increases epidermal protein deimination and drives human filaggrin breakdown[END_REF]. If all these works pinpoint at a barrier deficit when the skin is subjected to dry conditions, one should be cautious in extrapolating them to our study. Most of the works mentioned above were performed comparing much longer incubation times in different RH conditions than the two hours preconditioning time we used. In our case, preconditioning by the semi-dry airflow might have only led to the very first step of the above-mentioned changes: the increased water loss, the beginning of the dehydration of the upper layers of the stratum corneum and, as we show it, the release of cytokines/chemokines. Therefore, for VOCs the effect we observe would be driven by our dynamic experimental setup and would only reflect the effect of the two hours preconditioning in semi-dry conditions.

The fate of PM would be different but can also be explained by the mechanical characteristics of the skin under the different RH conditions. In that respect, the static experimental design we used for TS reflects the particulate nature of TS rather than that of its gaseous part since it induces a similar response than PM. Two mutually none exclusive aspects could explain the lower cytokine/chemokine induction observed upon preconditioning by the semi-dry airflow compared to the humid airflow. The first one is that, as mentioned above, the semi-dry airflow rapidly lead to dehydration of the upper layer of the stratum corneum and a scaly surface. These could have limited the contacts between PM and RHE and/or limited the diffusion of the chemical components absorbed to the surface of PM. On the other hand, preconditioning under humid E. Seurat et al.

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conditions might have favored these contacts. Using transmission and cryo-scanning electron microscopy, Warner et al. (2013) show that a four-hours treatment in an atmosphere saturated with water leads to a three-fold increase in the thickness of the stratum corneum with large pools of water accumulating in intercellular spaces where the lipid structures are disrupted. This study was performed in vivo, on the volar forearm of volunteers. With the weaker stratum corneum of RHE, we can assume that such an effect can occur when they are maintained under 90% RH. Not only would it explain better contact between PM and the upper cell layers, but the rupture of the stratum corneum barrier and the large pool of water might lead to an increased entry of PM. It would also explain a better diffusion of the metallic elements absorbed to the surface of PMm and, therefore, the stronger release of IL-1α, IL-8, and RANTES.

It is important to note that our conclusions are quite limited by the fact that the experiments were performed on a single batch of RHE which are known to show some inter-batch variability. A last limitation is due to the short period during which RHE can be used. This restricts the use of our system to the test of short exposure periods and precludes us from testing chronic like exposure. Still, the use of a 3D-skin model -a reconstructed human epidermis -enabled us to mimic the effects of close contact between pollutants and the skin. The experimental system we set up also allowed us to test realistic environmental pollutants. Not only are they realistic in their generation and, by extension, in their physicochemical composition but, also, in the way we delivered them to RHE, which reflects the real human dermal exposure route: an airliquid exposure resulting in the topical application of pollutants. Finally, the use of the

  construction and decoration labeling (decrees 19/04/2011 and 20/04/2012 that set the upper limit of VOCs to 20 g/L; European Union, 2010 and the French decree 2011-321 (March 23, 2011) related to the mandatory labeling of VOCs emission of building and decoration products. VOCsb (solvent-based) are from an alkyd paint (Brillant, Orion line, Guittet, France) manufactured before 2007, and thus before the above-mentioned decrees.

  innovative humidification station has proven to be suitable, and the conditions we tested E.Seurat et al. 25/39 are representative of real environmental conditions, since the 2 hours preconditioning is 515 sufficient to induce different reactions towards various environmental pollutants. 516 E. Seurat et al. 26/39ConclusionIn the course of this study, we tested various pollutants with different chemical compositions, applying them to the apical side of RHE and being particularly interested in the effect relative humidity has on the reaction to pollutants. The negative effect of pollutants on the cellular functionality is aggravated when RHE are preconditioned for two hours by a semi-dry airflow. Investigating several cytokines and chemokines, we showed that IL-1α, IL-6, IL-8, and RANTES are the cytokines/chemokines almost systematically induced by most pollutants. When RHE are preconditioned by a semidry/humid airflow before being subjected to pollutants, the release of IL-1α, IL-8, and RANTES falls into two groups. For VOCs, preconditioning by a semi-dry airflow exacerbates IL-1α, IL-8, and RANTES release, similar to what happens in the absence of pollutants, indicating a response driven by RH rather than by VOCs. On the other hand, for PM and TS, preconditioning by a semi-dry airflow leads to a decrease in the release of IL-1α, IL-8, and RANTES. Our interpretation is that the effect of RH on the skin structure primes over the chemical composition of the pollutants. It is the changes induced by the different humidity conditions -and our experimental design -that determine the outcome of the toxicological endpoint of a pollutant depending on its physical nature. E.Seurat et al.

Acknowledgments

The authors thank Dr. Gallic Beauchef for helpful discussions and comments. They also wish to thank Dr. Philippe Crouzet, Estium-Concept, for scientific writing services. E. Seurat et al.

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Declaration of interest

The authors declare no conflict of interest. Table 1 Summary of the main metallic and organic components of PMm (Cotonou, Benin -Cachon et al., 2014) and PMo (Dunkerque, France -Dergham et al., 2015).

CRediT authorship contribution statement

Tables

Chemicals PMm

(ng/m

Legends of the figures