Pan Dan

Patrick Menu
email: patrick.menu@univ-lorraine.fr

Roni Ue Ecot

Human-derived extracellular matrix from Wharton's jelly: An untapped substrate to build up a standardized and homogeneous coating for vascular engineering

Keywords: Biomimetic materials Vascular tissue engineering, Wharton's jelly, ECM (extracellular matrix), Coating

One of the outstanding goals in tissue engineering is to develop a natural coating surface which is easy to manipulate, effective for cell adhesion and fully biocompatible. The ideal surface would be derived from human tissue, perfectly controllable, and pathogen-free, thereby satisfying all of the standards of the health authorities. This paper reports an innovative approach to coating surfaces using a natural extracel-lular matrix (ECM) extracted from the Wharton's jelly (WJ) of the umbilical cord (referred to as WJ-ECM). We have shown by atomic force microscopy (AFM), that the deposition of WJ-ECM on surfaces is homogenous with a controllable thickness, and that this easily-prepared coating is appropriate for both the adhesion and proliferation of human mesenchymal stem cells and mature endothelial cells. Furthermore, under physiological shear stress conditions, a larger number of cells remained adhered to WJ-ECM than to a conventional coating such as collagen -a result supported by the higher expression of both integrins a2 and b1 in cells cultured on WJ-. Our data clearly show that Wharton's jelly is a hi hly promisin coatin for the desi n of human biocompatible surfaces in tissue engineering as well as in regenerative medicine.

Statement of Significance

Discovery and design of biomaterial surface are a hot spot in the tissue engineering field. Natural matrix is preferred to mimic native cell microenvironment but its use is limited due to poor resource availability. Moreover, current studies often use single or several components of natural polymers, which is not the case in human body. This paper reports a natural extracellular matrix with full components derived from healthy human tissue: Wharton's jelly of umbilical cord. econstitutin this matrix as a culture surface our easily-prepared coating provides superior biocompatibility for stem and mature cells. Furthermore, we observed improved cell performance on this coating under both static and dynamic condition. This novel human derived ECM would be a promising choice for regenerative medicine.

Introduction

The main goal of the tissue engineering triad is to create combi-nations of cells, scaffolds and environment (i.e. soluble mediators and/or mechanical stresses). For vascular tissue engineering, find-ing appropriate cells and scaffolds remain the two major challenges. Stem cells, and in particular, mesenchymal stem cells (MSCs), have emerged as the most promising source of suitable cells as a result of their good availability and ability to differentiate into vascular cells, as well as their immunosuppressive properties which are particularly interesting for allogeneic applications [1,[START_REF] Ankrum | Mesenchymal stem cells: immune evasive, not immune privileged[END_REF]. As to the scaffold, synthetic materials are most commonly used for engineered vascular grafts, but they are poor substrates for cells. Therefore, substantial effort has been made to develop new coatings, ranging from synthetic polymers to isolated natural extracel-lular matrix (ECM) to: (i) provide a solid base for cell anchorage to allow resistance to blood flow; (ii) favor endothelial differentiation of stem cells; and (iii) facilitate cell expansion to mimic the endothelial layer [START_REF] Guilak | Control of stem cell fate by physical interactions with the extracellular matrix[END_REF][START_REF] Stevens | Exploring and engineering the cell surface interface[END_REF]. Synthetic polymers have the advantage of being precisely tai-lored, and they can be combined with other biological interfaces, allowing for the construction of desired tissues [START_REF] Ringe | Regenerative medicine: selecting the right biological scaffold for tissue engineering[END_REF]. However, those which have been studied to date have often exhibited poor biocompatibility and/or immunogenicity, limiting their clinical use. In the case of ECM, decades of extensive research on its structure and biological influence on cell behavior and fate have shown that its physical properties (elasticity, stiffness, resilience to the cellular environment, etc.), specific chemical signals exhibited by its peptide epitopes, and the nanoscale topography of microenvironmental adhesive sites, are all relevant to the design of bio-mimetic surfaces [START_REF] Mark | Nanoscale engineering of biomimetic surfaces: cues from the extracellular matrix[END_REF]. Nonetheless, current studies usually exploit one or several purified ECM components to construct surfaces [START_REF] Gillette | Dynamic hydrogels: switching of 3D microenvironments using two-component naturally derived extracellular matrices[END_REF], a practice which does not faithfully replicate the native ECM entire environment [START_REF] Place | Complexity in biomaterials for tissue engineering[END_REF].

Complete ECM derived from human tissue could be an ideal source for natural matrices. In this context, tissues such as placenta and umbilical cord which are obtained from non-pathological medical procedures, could provide a good supply. For example, pla-centa has recently been treated using a chemical procedure (urea solubilization) to furnish a complex of ECM for tissue engineering [START_REF] Moore | Novel human-derived extracellular matrix induces in vitro and in vivo vascularization and inhibits fibrosis[END_REF]. Wharton's jelly of human umbilical cord contains various types of colla en and lycosamino lycan as well as rowth factors and/ or cytokines synthesized by mesenchymal stem cells, and thus may be viewed as a natural source for ECM-based coating [START_REF] Can | Concise review: human umbilical cord stroma with regard to the source of fetus-derived stem cells[END_REF]. Indeed, Hao et al. previously showed that mechanically-obtained from Wharton's jelly can support culture [START_REF] Hao | Culturing on Wharto ' j y x rac d ay m chyma m c c c hr gh p53 a d p16INK4a/pRb pathways[END_REF].

ere we describe a novel enzymatic method to isolate complete from Wharton's jelly (this material will be referred to hereafter as WJ-ECM), which boasts many benefits relative to existing procedures. The most important advantages of our coating are its human tissue origins and a method of preparation that does not involve chemicals which are toxic to cells. To test our material, we prepared, characterized, and determined the in vitro response to WJ-ECM of MSCs and endothelial cells, which are currently the two most-used cell sources in vascular tissue engineering, under both static and dynamic conditions. Our data show that WJ-ECM-derived coating enhanced proliferation of both human MSCs (hMSCs) and human umbilical vein endothelial cells (HUVECs). This new surface also exhibits improved cell resistance to flow under shear stress, a phenomenon which is of particular interest in vascular tissue engineering. The role of integrins in the possible mechanism underlying cell retention under shear stress was also investigated.

Methods

Preparation of WJ-ECM coating

Fresh human umbilical cords were obtained after full-term births with informed consent using the guidelines approved by the Cell Therapy Unit of the University Hospital Center (CHRU) of Nancy (Authorization number: TCG/11/R/011). Briefly, whole umbilical cord was washed 3 times with ank's balanced salt solu-tion (HBSS) and cut into 5-cm explants and Wharton's jelly was carefully isolated without taking umbilical cord membrane or ves-sels. The WJ of umbilical cord from 3 separate donors was then pooled prior to sample homogenization. The isolated WJ was fur-ther cut into 0.5-cm pieces using sterile surgical scissors. The WJ was then digested in trypsin (0.025%, Bioblock, 1158-0626, France) at 37 C for 24 h with magnetic stirring (ratio of wet WJ/trypsin was 1 g: 5 ml), the stirred suspensions were centrifuged at 16,000g for 10 min, and the collected supernatants were filtered (100 lm filter).

Fetal bovine serum (FBS) (Dutscher, S1900) was added into the solution (final concentration of 10%) to inhibit the trypsin reaction. The concentration of WJ (mg/ml) was calculated using lyophilized WJ tissues dissolved in final phosphate buffered saline (PBS). The obtained WJ-ECM solution was conserved at 4 C for further use. To coat culture surfaces, the freshly-obtained or conserved WJ-ECM was deposited onto glass coverslips which had been carefully pre-treated with SDS-HCl [START_REF] Berthelemy | Polyelectrolyte films boost progenitor cell differentiation into endothelium-like monolayers[END_REF]. The glass cover-slips coated with WJ-ECM were incubated for 12 h at 37 C, then washed twice with culture medium.

Characterization of the surface coating by AFM

Before each measurement, fresh samples were extensively rinsed with milli-Q water and then slowly and completely dried with nitrogen. AFM experiments were carried out using a MFP3D-BIO instrument (Asylum Research Technology, Atomic Force F&E GmbH, Mannheim, Germany). Silicon nitride cantilevers of conical shape were purchased from Olympus (OTR-400 PSA, Bruker-nano AXS, Palaiseau, France). The spring constants of the cantilevers measured using the thermal noise method were found to be 0.020-0.024 nN/nm. Experiments were performed both in air and in PBS buffer at room temperature. AFM images were recorded at room temperature (20 C) in contact mode. The applied force between the tip and the surface was carefully monitored and min-imized at approximately 0.2nN for experiments performed in aqueous medium. All images were collected with a resolution of 512 512 pixels at a scan rate of 1 z. he nanoindentation method was used to uantify the mechanical properties. Youn 's modulus of the thin films was calculated from force vs. indentation curves. Mechanical properties were obtained by recording a grid map of 32-by-32 force curves at different locations on the thin deposits over the 10 lm 10 lm surface area. The maximal load-ing force was 0.5 nN, the piezodrive was fixed to 1 lm and the approach rate was 1000 nm s 1 . he Youn 's modulus was esti-mated from the analysis of the approach curves according to the Sneddon model [START_REF] Sneddon | The relation between load and penetration in the axi-symmetric Boussinesq problem for a punch of arbitrary profile[END_REF].

where d is the indentation depth, m the Poisson coefficient, R the curvature radius of the AFM-tip apex and fBECC is the Bottom Effect Cone Correction function that takes into account the presence of substrate stiffness [START_REF] Gavara | Determination of the elastic moduli of thin samples and adherent cells using conical atomic force microscope tips[END_REF]. All the force curves were analyzed by means of an automatic Matlab algorithm described elsewhere [START_REF] Polyakov | Automated force volume image processing for biological samples[END_REF].

Biomolecular composition analysis

WJ-ECM solution (10mg/ml) was analyzed using a human angiogenesis array kit (R&D systems, ARY007, France) according to the manufacturer's protocol and the pixel density of detected spots was measured with ma e J software.

Culture of hMSC and HUVEC cells o isolate h

s from umbilical cord ( ) Wharton's jelly was sliced into -mm explants after removing the umbilical vessels and the umbilical outer layer membrane, and the slices were subse-quently attached and cultured in minimum essential medium Eaglealpha modification (a-MEM) (Lonza, BE12-169F) supple-mented with 10% FBS, Fungizone ! (100mg/ml, Fisher, 11520496), penicillin (100 IU/ml, Sigma, P0781) and L-glutamine (200 mM, Sigma, G7513) on culture plates. The medium was chan-ged every 3 days, and hMSC populations appeared as outgrowths from the UC fragments at day 6. After 15 days, the UC fragments were discarded, and the cells were passaged with trypsin and expanded until they reached sub-confluence. To isolate HUVECs, full length UC was taken, the umbilical vein was washed carefully with HBSS to remove residual blood, an appropriate volume of trypsin was injected into the vein, the trypsin was harvested after 20 min of incubation at 37 C, the suspension was centrifuged at 300g for 5 min, and finally, the pellet was suspended in endothelial basic medium (EBM TM -2, Lonza) supplemented with 10% FBS, Fun-gizone ! (100 mg/ml, Fisher, 11520496), penicillin (100 IU/ml, Sigma, P0781) and L-glutamine (200 mM, Sigma, G7513). Cells were cultivated on culture plates for 10 days until 90% confluence was reached.

Bioactivity of soluble factors in WJ-ECM

hMSCs at passage 4 were seeded onto 12-well plates at 3000 cells/cm 2 . One milliliter of culture medium a-MEM supple-mented with different percentages of WJ-ECM solution (2.5%, 10% and 40%, respectively) was added to different wells to observe its influence on cell proliferation. In order to ensure that the influence on cell proliferation was not due to FBS in the WJ-ECM solution but to active soluble factors in the solution, cells were seeded into dif-ferent culture mediums with or without FBS. The various culture conditions were: hMSCs cultured ±10% FBS (the usual concentra-tion in cell culture); WJ-ECM ± FBS, where (1) 40% of WJ-ECM solu-tion (containing 10% of FBS) corresponds to 10% FBS in the cell culture medium and (2) 10% of WJ-ECM solution (containing 2.5% of FBS) which corresponds to 2.5% FBS in the cell culture medium. (Note, as the 10% WJ-ECM contains 2.5% FBS, we also evaluated a 2.5% FBS control). After 5 days of cell culture, cells were stained with Live/Dead assay (Sigma-Aldrich, 04511, France) and observed using a fluorescence microscope.

Cell viability and proliferation

The viability of both hMSCs and HUVECs on different surfaces was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte trazolium bromide) assay. he assays were carried out as per the manufacture's protocol ( i ma-Aldrich, M5655). Briefly, cells were seeded onto a 24-well plate containing cover slides coated with WJ-ECM at different viscosities; collagen (0.1 mg/ml) was used as a positive control, and cover slips alone as a negative con-trol. Cells were seeded at a density of 5.10 3 cells/cm 2 . After 1, 3 and 5 days of culture, medium was removed, 10% MTT in fresh medium was added, and the cells were incubated for 4 h. The MTT solution was then carefully removed after incubation, and 200 ll of DMSO per well were added to dissolve the produced formazan, followed by a further incubation for 5 min. 100 ll of solution from each well were transferred to a new 96-well plate, and the absorption at 570 nm read using an automated plate reader (Varioskan Flash, Thermo scientific, France).

Cell proliferation by DNA quantification

In parallel, at each time point, total DNA of each sample was collected and quantified using a Hoechst 33258 fluorescent dye assay (Invitrogen, H3569, France). Briefly, cells cultivated on differ-ent surfaces were washed twice with PBS and the cell pellet was harvested, after which the total DNA content was extracted with two freeze (liquid nitrogen)-thaw (37C) cycles. Fluorescence was measured with a microplate fluorescence reader (Varioskan Flash, Thermo scientific, France) using equal volumes of cell lysates and Hoechst 33258 dye solution (100 lg ml 1 ). The number of viable cells per scaffold was calculated by extrapolation from a cal-ibration curve that was generated as described in Rago et al. [START_REF] Rago | DNA fluorometric assay in 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water[END_REF].

Endothelial differentiation of hMSCs

On passage 4, hMSCs were seeded at a density of 3000 cells/cm 2 onto glass coverslips coated with WJ-ECM or collagen or without coating. When cell confluence reached 40%, a commercial medium (EGM TM -2) which is used for EC differentiation was added into the culture plates. hMSCs were cultivated in EGM TM -2 for 14 days, with the medium being changed every 3 days. Protein and RNA were harvested for assessment of differentiation markers by Western-blot and quantitative real-time PCR, respectively.

Laminar shear stress assessment

To assess the cell adherence of both hMSCs and HUVECs under dynamic conditions, the cell nuclei were stained with Hoechst 33258 (Invitrogen, H3569) and the membrane with DiI (Invitrogen, D282), before being seeded onto coated glass coverslips. When the cell confluence reached 70%, the glass coverslips was assembled into a parallel-chamber flow system (Masterflex, USA). The shear stress that cells experienced was gradually increased to 20 dyn/ cm 2 , and applied for 12 h. After application of shear stress, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, France), photos were taken by fluorescence microscopy (Leica DMI 3000B). 10 randomly chosen areas were investigated before and after shear stress, and the resulting images analyzed using the soft-ware Image J to count cell numbers [17]. Cells were then stained using crystal violet (Sigma-Aldrich, France) for large-scale images. For gene expression assessment, RNA was harvested directly after shear stress.

Quantitative real-time PCR

Total RNA from cell samples was isolated using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from 100 ng total RNA using iScript Reverse Transcription Supermix (Bio-Rad). Quantitative Real-Time PCR (RT-PCR) was carried out using iQ TM SYBR ! Green Supermix (Bio-Rad) and primers designed by us for human cluster of differentiation (CD)31, vascular endothelial (VE)-cadherin, vascular endothelial growth factor receptor 2 (VEGFR2), integrin a2, a3, and b1 and the ribosomal protein S29 (RP 29) with the use of the StepOnePlus TM device (Thermofisher, France). Cycling parame-ters were 3 min at 95 C; 40 cycles of 30 s denaturation at 95 C and 1 min of elongation at the annealing temperature (60 C for CD31, VE-Cadherin, VEGFR2 and RP29, 54C for integrin a2, 56 C for integrin a3 and 58 C for integrin b1). Data were normal-ized using RP29 as a housekeeping gene. Analyses and fold differ-ences were determined using the comparative CT method. Fold change was calculated from the 44CT values with the formula 2 DDCT and data are reported relative to control values.

Statistical analysis

Results are presented as mean ± standard deviation. Statistical significance was determined by ANOVA followed by a t-test between each group; p-values less than 0.05 were considered as statistically significant.

Results

Extraction of WJ-from Wharton's jelly

As an initial step, we compared chemical, mechanical and enzy-matic procedures to extract the ECM from the WJ of the umbilical cord. These procedures included acetic acid, type II collagenase, PBS (mechanical) and trypsin-mediated methods. Acetic acid was chosen as it was previously used to isolate and purify collagen I [18], one of the WJ-ECM components. When applied to the WJ, one week was required to achieve complete digestion of the ECM, and the procedure failed to produce enough ECM to coat glass substrates. Type II collagenase is commonly used for isolation of MSCs from the connective tissue of umbilical cord [START_REF] Han | Optimization of human umbilical cord mesenchymal stem cell isolation and culture methods[END_REF], but atomic force microscopy (AFM) analysis showed that this procedure was too aggressive for ECM extraction, as no collagen fibers could be detected after digestion. Similarly, analysis by AFM showed that Wharton's jelly stirred in B did not enerate enou h products to coat glass substrates. Taken together, these data demonstrated that acetic acid, type II collagenase, and PBS were unsuitable for obtaining an appropriate coating surface (Fig. S1).

We next investigated whether trypsin is able to digest WJ-ECM, as it is a common enzyme used in routine cell culture. Dissociated WJ tissue was digested in 0.025% trypsin at 37 C for 24 h under magnetic stirring, the supernatant was collected after centrifuga-tion, and FBS was added to stop the trypsin reaction. This method allowed us to obtain a viscous solution, with the viscosity between different batches remaining stable (Fig. 1a). Twenty-four hour-digestion and further centrifugation removed cells or other debris in the WJ-ECM (Fig. 1b,c). The complete WJ-ECM solution was lyo-philized to obtain a dry powder, and the precise concentration of WJ-ECM in the solution was calculated according to this dry pow-der and original solution volume. The influence of FBS, trypsin and PBS in the mixed solution was also taken into account during the calculations.

Previous studies have demonstrated that WJ contains abundant growth factors, a beneficial feature as this would likely stimulate angiogenesis [START_REF] Whar | j y a a r rv r f p p d gr w h fac r ac a[END_REF]. Therefore, following the enzymatic extraction procedure, the biomolecular composition of the WJ-ECM was determined using a human angiogenesis array (Fig. S2). Out of the 55 proteins that were screened for, 16 were detected in WJ-ECM (Figs. 1d,S2). These included several essential growth factors for vascular cell differentiation such as vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2) and transforming growth factor-b1 (TGF-b1). According to the results, there was little variability between healthy donors. When WJ-ECM solution was used as a cell culture supplement, increased hMSCs proliferation was observed (Fig. S3). This result indicated that the soluble factors in WJ-ECM remained active.

Fabrication of WJ-ECM derived coating

We tested the coating capacity of WJ-ECM on positively and negatively charged glass coverslips. Our obtained AFM images showed that WJ-ECM extract could not coat an electrically-neutral glass coverslip, but could produce a non-continuous thin layer on a positively-charged surface. On the other hand, WJ-ECM reliably formed a continuous coating on a negatively-charged surface (Figs. S4S6).

To visualize the morphological features of the WJ-ECM coated surface, and in particular the thickness of the WJ-ECM-based coat-ing, different concentrations of WJ-ECM solution were pipetted onto negatively-charged glass coverslips and incubated for 12 h at 37 C before imaging by AFM. Depending on the concentration, WJ-ECM formed a homogeneous coating from 13 ± 5 nm to 74 ± 16 nm thick (Table 1 and Figs. S7S12). At 5 mg/ml, a thick-ness of 45 ± 12 nm was obtained (Fig. 2a,b), similar to that pro-duced by collagen (0.1mg/ml) which was used as positive control (Fig. 2c,d). As the stiffness of the coated material has a sig-nificant effect on cell adhesion, proliferation and/or differentiation [START_REF] Murphy | Materials as stem cell regulators[END_REF] the Youn 's modulus of the WJ-ECM was determined by the nanoindentation method usin A . echanical measurements performed with A revealed an inverse correlation between the coatin 's Young modulus and its thickness. At the lowest concentration (1 mg/ml), the stiffness of the coating reached a maximal value of 1.34 ± 0.19 MPa (n = 1024). At 5 mg/ml, the stiffness was 1.18 ± 0.15 MPa (n = 1024), a value below that obtained for a collagen coating used as positive control (2.04 ± 0.17 MPa (n = 1024)) (Fig. 2e). Such an increase in stiffness is likely due to a decrease in the water content of the layer, as well as greater crystalline packing of WJ-ECM components during the adsorption process.

Static cell adhesion and proliferation

To examine the behavior of the cells seeded on this surface, the viability of the hMSCs and HUVECs was examined. Human MSCs showed a significantly higher mitochondrial activity when cul-tured on 5-8mg/ml WJ-ECM than on glass coverslips alone (Fig. 3a). In the case of the HUVECs, no difference between WJ-ECM concentrations was observed. However, cellular mitochon-drial activity was also significantly enhanced when compared to the activity of cells cultured on glass coverslips (Fig. 3b).

We next evaluated at different time points the adhesion and proliferation of HUVECs and hMSCs on WJ-ECM coating, when applied at a concentration of 5 mg/ml. Commercially available col-lagen I at 0.1 mg/ml and glass coverslips were used as positive and negative controls, respectively. Cells cultured on a collagen-coated surface showed a slightly higher viability than those cultured on coverslips at all 3 time points (day 1, day 2 and day 3 for hMSCs, day 1, day 3 and day 7 for HUVECs, taking into account their differ-ent proliferation rates), but this difference was not statistically sig-nificant. Interestingly, however, the WJ-ECM coated surface afforded a significantly higher mitochondrial activity, when mea-sured at the third time point (Fig. 3c,d). Total DNA quantification was also performed to evaluate cell proliferation on different surfaces (glass, collagen and ECM coating). In agreement with the viability observation, the WJ-ECM-coated surface promoted cell proliferation both for hMSCs and HUVECs (Fig. 3e,f). The dif-ference in proliferation rates became significant between the glass and ECM coatings at days 3 and 7 for hMSCs and HUVECs, respectively.

Differentiation of hMSCs to endothelial-like cells

Published studies, as well as our previous data, suggest that cell culture surface and medium can contribute to variations in the differentiation capacity of hMSCs [1,[START_REF] Wingate | Compressive elasticity of three-dimensional nanofiber matrix directs mesenchymal stem cell differentiation to vascular cells with endothelial or smooth muscle cell markers[END_REF]. As VEGF has been detected in the WJ-ECM, we thought to further investigate the endothelial dif-ferentiation potential of hMSCs. For this, we treated hMSCs expanded on WJ-ECM with Endothelial Growth Medium-2 (EGM TM -2). Fourteen days after the induction of differentiation, Western-blot and RT-PCR revealed the expression of endothelial cell specific markers such as CD31, VEGFR2 and VE-Cadherin (Fig. 4a,b). These results confirmed that the functionalization of the cell culture surface is mandatory to induce differentiation of hMSCs, and show that the WJ-ECM coating is suitable for both the expansion and differentiation of hMSCs.

Cell retention under dynamic stimulation

Biomaterials in vascular tissue engineering must provide appro-priate cell anchorage to resist physiological shear stress. To evaluate cell retention on WJ-ECM, we expanded MSCs and HUVECs onto 3 different surfaces (WJ-ECM coating, collagen and glass coverslips), then subjected them to 12 h of laminar shear stress at 20dyn/cm 2 . Crystal violet staining showed that more cells remained on WJ-ECM coating than the other two surfaces after shear stress (Fig. 5a). Cells from both sheared and static samples were also stained for membranes and nuclei (Fig. 5b), images of 10 randomly chosen fields on these coatings were taken using a fluo-rescence microscope, and the cells were then counted using Image J Software [17]. Notably, the WJ-ECM-coated surface maintained 92 ± 2% of MSCs after shear stress, whereas the collagen-coated surface and control glass coverslips retained 78 ± 3% and 74 ± 5% of MSCs, respectively (Fig. 5c). The retention of HUVECs on these different surfaces after 12 h of shear stress showed a similar pat-tern: the WJ-ECM coated surface retained 92 ± 3% of the HUVECs whereas 75 ± 11% and 74 ± 13% were retained on collagen and on glass coverslips, respectively (Fig. 5c). These results clearly suggest that WJ-ECM coating allows a better cell anchorage than commercially-available collagen I coating or glass, with the conse-quence that WJ-ECM may be a suitable coating to build up a continuous layer to mimic the endothelium during vascular tissue engineering.

Gene expression responding to shear stress

Human MSCs and HUVECs are known to express a large range of integrin subunits [17, [START_REF] Ross | Integrins in mechanotransduction[END_REF][START_REF] Docheva | Human mesenchymal stem cells in contact with their environment: surface characteristics and the integrin system[END_REF]]. Here we selected 3 representative integrins (integrins a2, a3 and b1) [START_REF] Turner | Alpha2(VIII) collagen substrata enhance endothelial cell retention under acute shear stress flow via an alpha2beta1 integrin-dependent mechanism: an in vitro and in vivo study[END_REF][START_REF] Warstat | TGF-beta enhances the integrin alpha2beta1-mediated attachment of mesenchymal stem cells to type I collagen[END_REF], to evaluate their expression in cells cultivated on different surfaces in the presence or absence of shear stress. Under static conditions, hMSCs expressed significantly higher levels of integrin a2 on the WJ-ECM surface than on glass coverslips or collagen (Fig. 6a). Further-more, following 6 h of physiological shear stress, a significantly increased expression of integrins a2 and b1 on WJ-ECM was observed compared to the static condition on WJ-ECM (Fig. 6b).

Under the same dynamic conditions, a slight decrease of a2 and b1 expression was observed in hMSCs cultured on collagen, and a significant increase of a2 expression was observed in hMSCs on glass. As for the HUVECs, higher b1 expression was observed on WJ-ECM relative to the other two surfaces under static condition (Fig. 6c). Under shear stress, a significant increase in expression of integrins a2 and b1 was observed in HUVECs cultured on WJ-ECM. On collagen, this increase was only significant for b1 (Fig. 6d). Thus overall, both hMSCs and HUVECs exhibited a significant increase in expression of integrins a2 and b1 due to shear stress on WJ-ECM relative to collagen. In the case of integrin a3 expres-sion, no difference was detected under static culture conditions irrespective of the surface employed. (c) and ECs (d) on glass coverslips, collagen (0.1 mg/ml) and WJ-ECMcoated slips (5 mg/ml) was measured at three time points by MTT assay. The DNA content of each sample was also measured at those time points for MSCs (e) and ECs (f) (SD, n = 4, * p < 0.05 compared to glass coverslip surfaces). In all of the assays, the WJ-ECM coating at 5 mg/ml produced the best results in terms of both cell adhesion and proliferation.

Furthermore, a significant decrease in integrin a3 was observed after subjecting HUVECs cul-tured on all three surfaces to shear stress. Taken together, these results suggest that integrins a2 and b1 might be involved in help-ing cells cultured on WJ-ECM to resist shear stress.

In order to investigate the interaction between cells and WJ-ECM, we assessed the expression levels of mechanosensory com-plex of endothelia cells: CD31, VE-cadherin and VEGFR2 in HUVECs on this surface, and compared them with those obtained on colla-gen and glass coverslips under both static and dynamic conditions. Gratifyingly, higher levels of CD31, VE-cadherin and VEGFR2 were detected in HUVECs cultured on WJ-ECM than from cells cultured on collagen or glass coverslips (Fig. 6e).

Even more interestingly, dynamic conditions (20 dyn/cm 2 lam-inar shearing for 6 h), increased expression of these genes in cells cultured on collagen, but significantly decreased expression of these genes in cells culture on glass (Fig. 6f). Therefore, dynamic conditions enhance expression of the mechanosensory complex in cells grown on both collagen and WJ-ECM.

Discussion

Cardiovascular diseases, particularly atherosclerotic coronary artery diseases are still the leading cause of mortality the world over [START_REF] Mozaffarian | American heart association statistics committee and stroke statistics subcommittee, heart disease and stroke statistics-2016 update: a report from the American heart association[END_REF]. Tissue engineered vascular grafts which incorporate cells into a biodegradable scaffold, have emerged as a promising alternative for those patients who lack appropriate autologous graft candidates [START_REF] Benrashid | Tissue engineered vascular grafts: origins, development, and current strategies for clinical application[END_REF]. The main hurdle in small-diameter (<6 mm) vascular tissue engineering is the thrombogenicity of newly designed grafts. Studies aimed at resolving this challenge have to date focused on investigating cell sources and discovering new scaf-fold/coating that promote full cellularization. MSCs are now considered as the most promising source for vascular tissue engineering due to their excellent availability, ability to differentiate into smooth muscle cells and endothelial cells under appropri-ate stimulation, and their immunosuppressive properties [START_REF] Seifu | Small-diameter vascular tissue engineering[END_REF][START_REF] Krawiec | Adult stem cell-based tissue engineered blood vessels: a review[END_REF]. On the other hand, mature endothelial cells are still the first choice for the purpose of mimicking the native environment [START_REF] Moore | Novel human-derived extracellular matrix induces in vitro and in vivo vascularization and inhibits fibrosis[END_REF]. Only grafts that are well cellularized or which can recruit circulating cells, can avoid long-term graft failure [START_REF] Zhao | The development of a tissue-engineered artery using decellularized scaffold and autologous ovine mesenchymal stem cells[END_REF][START_REF] Koobatian | Successful endothelialization and remodeling of a cell-free small-diameter arterial graft in a large animal model[END_REF]. To this end, new synthetic and natural isolated scaffolds/coatings have been exten-sively studied. Unlike traditional approaches which isolate one or few components of tissue extracellular matrix, the full comple-ment of ECM components is preferred in order to mimic the native cell environment. However, non-pathological human source is often limited. We describe in detail here a novel full ECM derived from healthy human Wharton's jelly (WJ-ECM).

The isolation of ECM from human tissue can be challenging in terms of restoring all of the functional components. Mechanical, chemical and enzymatic methods are most typically employed in ECM isolation. For example, ECM has been extracted from human placenta by chemical procedures and shown to contain various functional cytokines [START_REF] Moore | Novel human-derived extracellular matrix induces in vitro and in vivo vascularization and inhibits fibrosis[END_REF]. In this study, WJ tissue simply stirred in PBS failed to generate enough product to form a coating, while ECM derived from tissue dissolved in acetic acid was also unsatis-factory. Furthermore, this chemical treatment is a time-consuming procedure, and is typically used for collagen I isolation but not for obtaining full ECM. Here we choose to isolate complete ECM using enzymatic methods to simplify the isolation procedure, and more importantly, to maintain the ECM-containing tissue in a relatively physiological condition. We next compared collagenase II with trypsin, as both are routinely used in cell culture. Collagenase II generates a viscous solution, but which was unable to form a coating for in vitro cell culture, whereas trypsin readily yielded a vis-cous solution from human Wharton's jelly which was easily coated onto surfaces. Furthermore, abundant biological molecules such as VEGF, FGF-2, TGF-b1, endothelin-1, etc., which are consid-ered important in vascular function, were detected in the solution [START_REF] Briquez | Extracellular matrix and growth factor engineering for controlled angiogenesis in regenerative medicine[END_REF]. As we used a semi-quantitative method to measure the growth factors and cytokines in the current study which allowed us to detect a large panel of factors, further quantitative assays and characterization of WJ-ECM will be required to fully character-ize its composition in solution. These active growth factors and cytokines may allow WJ-ECM solution to be utilized in other appli-cations, such as cell culture supplement and the formation of a gel for cell assays.

For studies in vitro, biomaterials are typically coated onto glass coverslips in order to obtain a surface with appropriate thickness. ECM proteins such as collagen and fibronectin can form a coating directly on glass coverslips and polystyrene-based culture surfaces [START_REF] Sherratt | Substrate chemistry influences the morphology and biological function of adsorbed extracellular matrix assemblies[END_REF], and even polyelectrolyte films exhibiting a certain charge can coat surfaces [START_REF] Berthelemy | Polyelectrolyte films boost progenitor cell differentiation into endothelium-like monolayers[END_REF]. Inspired by this idea, we tested the influence of different surface charge on the coating efficiency. AFM results revealed that negatively-charged glass coverslips can facilitate WJ-ECM coating. Furthermore, compared to commercial collagen coating, WJ-ECM yielded a homogenous surface without collagen fibers. The thickness and stiffness of WJ-ECM coating depended on ECM concentration.

It has been previously shown that modified ECM coating can enhance cell adhesion and proliferation [START_REF] O' Va | Kava agh rac R Wa h c gh mpar g h d helialisation of extracellular matrix bioscaffolds with coated synthetic vascular graft materials[END_REF], probably due to several features such as its thickness and stiffness, and the incorporation of biological molecules [START_REF] Mark | Nanoscale engineering of biomimetic surfaces: cues from the extracellular matrix[END_REF]. In this study, the collagen-modified surface demonstrated higher cell adhesion and proliferation than the glass surface, but the WJ-ECM coating exhib-ited the highest cell attachment and proliferation with both hMSCs and HUVECs. Because the WJ-ECM is derived from human tissue, and the isolation procedure is completely physiological, WJ-ECM did not show any toxicity to cells. These features should make WJ-ECM a versatile tool in various type of tissue engineering to support cell cultures. Furthermore, WJ-ECM was capable of facili-tating the differentiation of hMSCs into endothelial-like cells by expressing endothelial cell specific markers. This approach could allow the construction of a tissue engineered vessel using a single cell source [START_REF] Zhao | The development of a tissue-engineered artery using decellularized scaffold and autologous ovine mesenchymal stem cells[END_REF].

In native blood vessels, the endothelium is exposed to stable shear stress that is generated by the flow of blood. Therefore, tissue engineered vascular grafts must support cell attachment under physiological shear stress. Surface components, and in particular, topography and stiffness, can influence cell attachment under dynamic condition [START_REF] Docheva | Human mesenchymal stem cells in contact with their environment: surface characteristics and the integrin system[END_REF]. A major limitation of current vascular tissue engineering is the low number of endothelial cells (ECs) or EC-replacing cells remaining after exposure to fluid shear stress representative of blood flow. Nonetheless, studies with modified substrates such as collagen coating have shown an amelioration in cell retention under shear stress conditions [START_REF] Doyle | Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractilitydependent adhesions[END_REF][START_REF] Han | Cell adhesion on zein films under shear stress field[END_REF]. However, our results have shown that a collagen coating failed to improve cell resistance to laminar shear stress (although this behavior might be attributed to the use of a relatively lower concentration of collagen (0.1 mg/ml) relative to previous studies), whereas WJ-ECM significantly retained cells exposed to 6 h of shear stress. The underlying mechanism of the increased cell retention might be related to the stiffness of WJ-ECM surface, adhesive elements incorporated in the coating, and an interaction between cells, cul-ture surface and shear stress [START_REF] Gong | Physiological pulsatile flow culture conditions to generate functional endothelium on a sulfated silk fibroin nanofibrous scaffold[END_REF]. Among the molecules involved in cell retention, integrins are a large family of transmembrane, heterodimeric cell-surface molecules, believed to be the principle adhesion factors for cells to attach to their surfaces. They establish cytoskeletal dynamics [START_REF] Ross | Integrins in mechanotransduction[END_REF]. Integrins can thus be considered as cell bonds with a range of ligands existing on ECM, and regulate important indicators of the interaction between cells, ECM and dynamic forces. It has been reported that integrins of ECs were responsible for cell retention under shear stress [START_REF] Turner | Alpha2(VIII) collagen substrata enhance endothelial cell retention under acute shear stress flow via an alpha2beta1 integrin-dependent mechanism: an in vitro and in vivo study[END_REF], as well as improved retention of MSCs with concomitant upregulation of integrins [START_REF] Mcilhenny | Linear shear conditioning improves vascular graft retention of adipose-derived stem cells by upregulation of the alpha5beta1 integrin[END_REF]. Our results show that both hMSCs and HUVECs exhibit a significant increase in expression of integrins a2 and b1 due to shear stress. On the other hand, once shear stress is applied to cells seeded on a defined support, the cells transmit a signal from their apical surface through the cytoskeleton to points of attachment, in order to foster cell-matrix adhesions [START_REF] Watt | Role of the extracellular matrix in regulating stem cell fate[END_REF]. It has been proposed that CD31, VE-cadherin and VEGFR2 serve as a mechanosensory complex for the attachment of ECs in response to shear stress [START_REF] Tzima | A mechanosensory complex that mediates the endothelial cell response to fluid shear stress[END_REF]. Indeed, these three junctional adhesion mole-cules play crucial roles in cell-cell and cell-matrix attachment. In addition, cell on different surfaces has been reported to modify the expression level of EC functional markers [START_REF] Chlupac | The gene expression of human endothelial cells is modulated by subendothelial extracellular matrix proteins: short-term response to laminar shear stress[END_REF]. Our results have revealed that the expression of the mechanosensory complex by ECs cultured on WJ-ECM coating was relatively high and was maintained with or without shear stress, which may explain the retention of the cells on the coating. On the other hand, HUVECs cultured on collagen under shear stress increased expres-sion of mechanosensory complex, whereas expression of mechanosensory complex by cells grown on glass coverslips decreased significantly. Taken together, these results indicate that the WJ-ECM coating may find application in cell culture under both static and dynamic conditions.

Conclusion

In summary, we have demonstrated that our new, easily-prepared WJ-ECM coating is appropriate for both the adhesion and proliferation of the two cell types most commonly employed in vascular tissue engineering, human mesenchymal stem cells and mature endothelial cells. Furthermore, under conditions of physiological shear stress, a larger number of cells remained adherent to WJ-ECM than onto a conventional coating such as col-lagen a result supported by a higher expression of both integrins a2 and b1 in hMSCs and HUVECs cultured on WJ-ECM. Thus, over-all our data clearly show that Wharton's jelly derived-ECM is a highly promising coating for cell culture surfaces in tissue engi-neering and regenerative medicine.

Fig. 1 .

 1 Fig. 1. Characterization of WJ-ECM solution. (a) The viscosity of WJ-ECM depended on its concentration. (b-c) Optical microscopic images showing the appearance of commercial collagen (0.1 mg/ml) (b) and WJ-ECM (5 mg/ml) (c) deposited on glass coverslips. (d) Cytokine analysis of WJ-ECM solution (10 mg/ml) was performed using a human angiogenesis array, and data were normalized on a scale ranging from negative control values (0%) to positive control values (100%). Data are representative of 3 independent batches, where each batch contained WJ tissue collected from 3 different healthy donors.

Fig. 2 .

 2 Fig. 2. Characterization of WJ-ECM derived coating. (a) Atomic force microscopy image of WJ-ECM at 5 mg/ml coated onto negatively-charged coverslips showing formation of a homogenous surface. (b) Cross-section of WJ-ECM coating, revealing a measured thickness of 45 ± 12 nm and an even distribution. (c) Commercial collagen (0.1 mg/ml) coating which served as a positive control. Collagen fibers are observed and they showed a random distribution. (d) The average thickness of the collagen coating was 37 ± 15 nm. (e) Youn 's modulus of colla en coatin at 0.1 m /ml and WJ-ECM coating at 5 mg/ml.

Fig. 3 .

 3 Fig. 3. Cell behavior on different surfaces. Cells were expanded on different coating surfaces formed from various concentrations of WJ-ECM. Cell adhesion was evaluated by MTT assay for mesenchymal stem cells (MSCs) after 72 h (a) and for endothelial cells (ECs) after 9 days (b). A single factor ANOVA test allowed rejection of the null hypothesis for all assays and a Tukey test between all groups was performed (SD, n = 4, * p < 0.05). Cell proliferation of MSCs (c) and ECs (d) on glass coverslips, collagen (0.1 mg/ml) and WJ-ECMcoated slips (5 mg/ml) was measured at three time points by MTT assay. The DNA content of each sample was also measured at those time points for MSCs (e) and ECs (f) (SD, n

Fig. 4 .

 4 Fig. 4. Mesenchymal stem cells differentiate into an endothelial-like cell lineage. MSCs cultured on 3 different surfaces were treated with EGM TM -2 for 14 days. (a) Endothelial specific gene expression was normalized to the house keeping gene RP29 and then normalized to positive HUVECs. (b) Expression of endothelial specific markers by Westernblot after EGM TM -2 treatment. HUVECs were taken as a positive control, while untreated MSCs cultured on glass served as a negative control.

Fig. 5 .

 5 Fig. 5. Cell behavior under dynamic conditions. (a) and (b), Confluent cells cultured on 3 different surfaces (Static) were subjected to shear stress at 20 dyn/cm 2 for 12 h (Dynamic). (a) Cells were stained with crystal violet. Scale bar, 1 mm. (b) Fluorescence microscopy images in which the cell membrane was stained with DiI (red) and the nucleus was stained with Hoechst 33258 (blue). Scale bar, 100 lm. (c) Cell retention after 12 h of shear stress (n = 4. Statistical significance, indicated by (*), was determined with a oneway ANOVA followed by Tukey pairwise comparisons between all groups, a = 0.05). Cell retention was calculated by counting cell numbers before and after shear stress. Cells were counted by Hoechst staining on 10 random areas (0.721 0.54 mm/area) on each culture surface. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6 . 2 ;

 62 Fig. 6. Gene expression of MSCs and HUVECs cultured on different surfaces under static and dynamic conditions. Expression of integrins in hMSCs (a) and HUVECs (c) under static culture on glass coverslips, collagen and WJ-ECM. Expression levels are normalized to cells on glass coverslips (blue bars, * p < 0.05, ** p < 0.01). Changes in integrin expression levels after 6 h of shear stress at 20 dyn/cm 2 for hMSCs (b) and HUVECs (d); expression levels were normalized to static conditions on glass coverslips, collagen and WJ-ECM, respectively. # indicates significant changes ( # p < 0.05, ## p < 0.01) compared to static expression levels. (e) Expression of mechanosensory complex proteins (CD31, VE-cadherin and VEGFR2) in HUVECs under static culture; mRNA levels are normalized to those in cells grown on glass coverslips (blue bars, * p < 0.05). (f) Changes in the mechanosensory complex after 6 h exposure to shear stress at 20 dyn/cm 2 ; statistical analysis was performed as for integrins, where # indicates significant changes ( # p < 0.05, ## p < 0.01) compared to static expression levels. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Acknowledgements

The authors acknowledge the support of r. Jalal Bacharouche and the platform pectroscopy and icroscopy of nterfaces ( Jean Barriol nstitute ancy) where A experiments were performed. We would like to thank the research federation 0 Bioin e nierie ole culaire, Cellulaire et he rapeuti ue (Nancy, France) and China Scholarship Council (China). We would like to thank Miss Huiyi Zhang for her assistance with the prepara-tion of WJ-ECM coating. We would also like to thank Prof. Kira J. Weissman for careful proof-reading of this manuscript. Appendix A. Supplementary data: Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2016.10. 018.


Changhwan Oh

Marc Philippe

Denise Pons

Anais A Boura

A new type of homoxylous Mesozoic wood from Asia, with palaeogeographical and palaeoecological implications

come

Changhwan OH 1* , Anaïs BOURA 2 , Marc PHILIPPE 3 , Denise PONS 2 1 Department of Biological Sciences, Chonbuk National University, Jeonju, 561-756, Republic of Korea, bluegaia@jbnu.ac.kr 2 Université Pierre & Marie Curie (UPMC), UMR-CNRS7207, CR2P, MNHN/UPMC/CNRS, Bâtiment de Géologie, CP48, 57 rue Cuvier 75231 Paris Cedex 05, France 3 UMR5276 and Université Lyon-1, Campus de la Doua, F69622 Villeurbanne Cedex, France

In 1936, Shimakura reported on a fossil wood taxon named Dadoxylon (Araucarioxylon) japonicum Shimakura, on the basis of what he considered as a typical anatomy for the Araucariaceae. However it has a very unique feature, unlike other araucarian type fossil woods. Usually the radial tracheid pits of araucarian fossil wood are contiguous and alternate when they are bi-to multiserate (Fig. 1B). However, D. japonicum has long chains of opposite to sub-opposite square pits on its tracheid radial walls (Fig. 1C). We named this type of tracheid radial pitting the 'japonicum-type'. In some cases opposite or sub-opposite radial tracheid pits could be found from araucarian fossil wood, however these are very rare and local. In woods with abietinean radial pitting biseriate pits are usually opposite, but not contiguous and not square in out line (Fig. 1A). These peculiar woods were previously published as Dadoxylon Unger, Protopodocarpoxylon Eckhold or Brachyoxylon Hollick et Jeffrey, however we think it could be established a new genus for these woods with the japonicum-type of radial tracheid pitting.

Fossil woods which have the japonicum-type of radial tracheid pitting were surveyed bibliographically of hundreds of specimens in several collections were reappraised as well. Interestingly these woods are well constrained in time and space, viz. to eastern Asia, from Thailand to Japan, and to the Middle Jurassic to Early Cretaceous interval (Table 1, Fig. 2). This new type of Mesozoic wood can be considered as an endemic taxon to Eastern Asia. The main distribution of the fossil woods is in Indochina region of SE Asia and partly in Lhasa of Central Tibet and in SW Japan. On a present day Lhasa is far away from Indochina, but it belongs to a block that split relatively late from Gondwanaland and get accreted to Asia Mainland at least during the Late Jurassic. By the Early Cretaceous the palaeolatitudes of Indochina and Lhasa were similar (Metcalfe, 2013), and these tectonic units were probably neighbors for a while before to be torn away during the Himalayan collision (Fig. 2). In Japan the exact origin of the Torinosu Group of the Chichibu Terrane where the japonicum-type woods occur is unknown yet. However, by the Palaeozoic the Chichibu Terrane was probably at low latitude (Aitchinson et al., 1991). From Lhasa through Indochina and to SW Japan, the distribution of the japonicum-type woods stretches along what was during the Jurassic-Early Cretaceous a tectonically active margin, probably lined by a system of coastal cordilleras. From various evidences, including palaeofloras, climate on the southern side of these cordilleras was probably warm-temperate to sub-tropical and wet (Oh et al., 2011). So the spatio-temporally limited distribution of the japonicum-type wood suggests that this morphogenus might be of palaeogeographical and palaeoclimatological significance. Figure 1A-C. The typical abietinean and araucarian types radial pitting compared to the japonicum-type. Fig. 1A is Taxodioxylon pseudoalbertense from Heilongjiang, China (no. JNU71122). Fig. 1B is Agathoxylon togeumense from Gurye, Korea (no. GR007). Fig. 1C is Dadoxylon japonicum from Yatsuji, SW Japan (no. 53325).

Table 1. Spatio-temporal distribution of the japonicum-type fossil woods occurrences.

Figure 2 .

 2 Figure 2. The distribution of the japonicum-type fossil woods at the Early Cretaceous on Indochina region and Lhasa (black dots are the japonicum-type fossil wood; modified from Metcalfe, 2013). Japanese data not plotted as their position at this time is uncertain.