Tiffanie Lescure
email: tiffanielescure@gmail.com

Catherine Joulian
email: c.joulian@brgm.fr

Clément Charles
email: clement_cm45@yahoo.fr

Taoikal Ben
email: taoikalali@hotmail.fr

Ali Saanda

Mickael Charron
email: m.charron@brgm.fr

Dominique Breeze
email: d.breeze@brgm.fr

Pascale Bauda
email: pascale.bauda@univ-lorraine.fr

Fabienne Battaglia-Brunet

Simple or complex organic substrates inhibit arsenite oxidation and aioA gene expression in two β-Proteobacteria strains

Keywords: Betaproteobacteria strains, organic substrates, arsenite oxidation, aioA gene expression

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

Introduction

High concentrations of the toxic metalloid arsenic (As) in soils may be the result of pollution from mining, industrial activities or agricultural practices, or they may be due to the geochemical background. In the case of mining and metallurgical activities, As is released as a by-product in the waste (tailings) or from drainage galleries [START_REF] Casiot | Bacterial Immobilization and Oxidation of Arsenic in Acid Mine Drainage (Carnoulès Creek, France)[END_REF].

Biological arsenite (AsIII) oxidation processes, coupled to iron oxidation and precipitation, can be used advantageously for effective bioremediation of mine water [START_REF] Casiot | Bacterial Immobilization and Oxidation of Arsenic in Acid Mine Drainage (Carnoulès Creek, France)[END_REF][START_REF] Battaglia-Brunet | A simple biogeochemical process removing arsenic from a mine drainage water[END_REF] and, where soils are concerned, to attenuate the environment toxicity, since arsenate (AsV) is less toxic than AsIII. This oxidizing reaction is of significant ecological importance as it leads to the stabilization of arsenic in the environment since AsV is more readily adsorbed than AsIII by carrier phases such as iron or manganese oxides [START_REF] Fuller | Surface chemistry of ferrihydrite: Part 2. Kinetics of arsenate adsorption and coprecipitation[END_REF][START_REF] Smedley | A review of the source, behaviour and distribution of arsenic in natural waters[END_REF].

Bacteria able to oxidize AsIII and reduce AsV have been found in many terrestrial and aquatic environments [5][START_REF] Campos | Pseudomonas arsenicoxydans sp nov., an arsenite-oxidizing strain isolated from the Atacama desert[END_REF][START_REF] Duquesne | Arsenite oxidation by a chemoautotrophic moderately acidophilic Thiomonas sp. : from the strain isolation to the gene study[END_REF][START_REF] Hoeft | Dissimilatory Arsenate Reduction with Sulfide as Electron Donor: Experiments with Mono Lake Water and Isolation of Strain MLMS-1, a Chemoautotrophic Arsenate Respirer[END_REF][START_REF] Quéméneur | Population structure and abundance of arsenite-oxidizing bacteria along an arsenic pollution gradient in waters of the upper isle River Basin, France[END_REF][START_REF] Cordi | Determination of physiological, taxonomic, and molecular characteristics of a cultivable arsenic-resistant bacterial community[END_REF]. Some bacteria possess genes allowing oxidation as well as genes conferring the ability to reduce AsV to AsIII via the Ars resistance system. Although this AsV-reduction detoxification intracellular reaction is energy consuming, it is widely found in microbial communities because AsIII thus obtained is excreted out of the cell, or is sequestered as reduced glutathione (GSH) or other thiol conjugates, for example in yeast [START_REF] Rosen | Biochemistry of As detoxification[END_REF].

Another way in which bacteria derive energy from arsenic is via the dissimilatory reduction of AsV into AsIII by anaerobic respiration via the Arr system, using AsV as a final electron acceptor [START_REF] Silver | Genes and enzymes involved in bacterial oxidation and reduction of inorganic arsenic[END_REF]. The AsIII oxidation function in bacteria is encoded by the aio operon [START_REF] Lett | Unified nomenclature for genes involved in prokaryotic aerobic arsenite oxidation[END_REF], which comprises up to six genes (aioB, aioA, aioS, aioR, aioX and aioE). Two of them encode the two AsIII oxidase enzyme subunits: aioB, which encodes the small subunit containing the Rieske [2Fe-2S] cluster; and aioA, which encodes the large subunit containing a catalytic molybdopterin and a cluster [3Fe-4S]. These two genes were originally identified and isolated from two β-proteobacteria: Alcaligenes faecalis [14] and Herminiimonas arsenicoxydans 4 [START_REF] Muller | Arsenite Oxidase aox Genes from a Metal-Resistant-Proteobacterium[END_REF]. The other four aio genes correspond to the following functions: aioS encodes a histidine kinase sensor, aioR a transcriptional regulator, aioX an oxyanions-ligand protein, and aioE an AsIII oxidation electron transporter [START_REF] Wang | An Oxidoreductase AioE is Responsible for Bacterial Arsenite Oxidation and Resistance[END_REF]. Santini and van den Hoven [START_REF] Santini | Molybdenum-Containing Arsenite Oxidase of the Chemolithoautotrophic Arsenite Oxidizer NT-26[END_REF] studied a chemolithoautotrophic bacterium, strain NT26 that derives energy from AsIII oxidation but is not able to grow on minimal medium in the presence of arsenic when its aioA gene is inactivated. This experiment demonstrated the use of arsenic as an energy source by some bacteria. Many other bacteria have been identified as carrying genes encoding AsIII oxidase [START_REF] Oremland | Anaerobic Oxidation of Arsenite in Mono Lake Water and by a Facultative, Arsenite-Oxidizing Chemoautotroph, Strain MLHE-1[END_REF][START_REF] Santini | New Arsenite-Oxidizing Bacteria Isolated from Australian Gold Mining Environments-Phylogenetic Relationships[END_REF]. This is the case for the "Thiomonas (T.) arsenivorans" strain, a chemolithoautotrophic, acid-tolerant and mobile Gram-negative rod [5,[START_REF] Battaglia-Brunet | Proposal that the arsenite-oxidizing organisms, Thiomonas cuprina and 'Thiomonas arsenivorans' be reclassified as strains of Thiomonas delicata, and emended description of Thiomonas delicata[END_REF]. It has been proven elsewhere that this bacterium, by oxidizing AsIII into AsV, can both create energy and detoxify the environment [5]. Some publications suggest a very significant effect of organic substrates on bacterial AsIII oxidation in the presence of oxygen. Challan-Belval et al. [START_REF] Challan-Belval | Enhancing pozzolana colonization by As(III)-oxidizing bacteria for bioremediation purposes[END_REF] have shown that addition of yeast extract substantially reduces the rate of AsIII oxidation by bacteria organized in biofilms. Bachate et al. [START_REF] Bachate | Oxidation of arsenite by two betaproteobacteria isolated from soil[END_REF] observed a decrease in enzymatic AsIIIoxidase activity of two pure strains as the total concentration of organic substrates (acetate and yeast extract) in the growth medium increased. Lescure et al. [START_REF] Lescure | Influence of organic matters on AsIII oxidation by the microflora of polluted soils[END_REF] observed a negative effect of organic substrates input, with concentrations from 0.08 to 0.4 g of carbon/L, corresponding to 0.2 to 1 g/L yeast extract, on the AsIII-oxidation rate constant of soil microbial communities. It therefore appears that organic substrates slow down the transformation of AsIII into AsV by bacteria in aerated conditions, and may decrease the retention of As in the environment. Nandre et al. [START_REF] Nandre | Enhanced Detoxification of Arsenic Under Carbon Starvation: A New Insight into Microbial Arsenic Physiology[END_REF] showed that the presence of acetate in the growth medium induced a lower aioA gene expression in diverse bacterial strains isolated from tannery effluent and soils. Organic substrates may inhibit the aio encoded oxidation processes, because bacteria would preferentially metabolize simple or complex organic substrates providing more energy than AsIII. Organic substrates may also induce a decrease 5 in AsIII bioavailability by complexation reactions [START_REF] Spuches | Thermodynamics of the AsIII-thiol interaction: arsenite and monomethylarsenite complexes with glutathione, dihydroglucoic acid and other thiol ligants[END_REF]. Because of these complexation reactions, if AsIII oxidation was mainly or exclusively a detoxification mechanism, the presence of a high concentration of organic substrate may have a protective effect against AsIII, inducing an attenuation of the detoxification process.

However, to date, the influence of the concentration and nature of different organic substrates on biological AsIII oxidation has not been studied systematically.

The objective of this work was to study the influence of two types of organic substrates, complex or simple organic substrates, on the ability to oxidize AsIII of two β-proteobacteria strains presenting different metabolisms. One of these, a strain of Thiomonas delicata referred to as "T. arsenivorans" -uses AsIII as energetic substrate; the other, Herminiimonas (H.) arsenicoxydans, does not. The hypothesis to be tested trough this experiment was that bacterial AsIII oxidation could be inhibited differently by organic substrates when AsIII is used or not as energy source. A common complex organic substrate was chosen: yeast extract, a reference complex organic substrate in laboratory studies that can promote the growth of both strains. In parallel, because the strains do not use any common simple organic substrate, two simple organic substrates were selected; acetate for H. arsenicoxydans and succinate for "T. arsenivorans". Two sets of experiments were carried out. First, AsIII oxidation and aioA gene expression were studied in batch growth experiments with a relatively high concentration of arsenic allowing strain growth with AsIII as sole energy source: 75 mg.L -1 (or 1 mM) AsIII. Then, AsIII oxidation kinetics were determined at an initial AsIII concentration more compatible with the concentrations commonly found in the pore water of polluted soils (0.5-2 mg.L -1 AsIII, [START_REF] Battaglia-Brunet | Monitoring of arsenic bio-oxidation and bio-redution activities in highly As-polluted soils during a phytostabilization field experiment (Phytoperf project)[END_REF]). 6

Materials and methods

2.1.Bacterial strains

Two AsIII-oxidizing microorganisms carrying an aioA gene were selected as models;"Thiomonas arsenivorans" (DSM 16361), a mixotrophic and optional autotrophic strain able to grow on AsIII and using only the AsIII oxidation reaction as an energy source;

Herminiimonas arsenicoxydans (DSM 17148), a heterotrophic strain perfectly characterized [START_REF] Muller | Herminiimonas arsenicoxydans sp. nov., a metalloresistant bacterium[END_REF].

These two strains belong to the subdivision of β-Proteobacteria. "T. arsenivorans" was isolated by BRGM [5]. H. arsenicoxydans [START_REF] Weeger | Oxidation of arsenite to arsenate by a bacterium isolated from an aquatic environment[END_REF] was kindly provided for this study by the laboratory of Molecular Genetics, Genomics, Microbiology (GMGM) of Louis Pasteur University (Strasbourg, France), where the strain was isolated.

Culture media

"T. arsenivorans" was maintained in minimal CAsO1 medium [START_REF] Battaglia-Brunet | An arsenic(III)-oxidizing bacterial population: selection, characterisation, and performance in reactors[END_REF] with 100 mg.L -1 AsIII, and H. arsenicoxydans, which is a heterotrophic bacterium, in the Luria-Bertani (LB) medium, then sub-cultured in CAsO1 medium amended with 1 g.L -1 yeast extract before each experiment.

The CAsO1 liquid medium is a mixture of 500 mL of each of two solutions. Solution A contains 0.5 g KH 2 PO 4 , 0.5 g K 2 HPO 4 , 0.5 g NaCl, 0.05 g (NH 4 ) 2 SO 4 and 1 mL of trace elements in solution [START_REF] Battaglia-Brunet | An arsenic(III)-oxidizing bacterial population: selection, characterisation, and performance in reactors[END_REF]. The pH of this solution was adjusted to 6.0 before autoclaving for subsequent growth of "T. arsenivorans", and to 7.2 for growth of H. arsenicoxydans. Solution B (500 mL) contains 0.1 g CaCl 2 and 0.1 g MgSO 4 . Both solutions were autoclaved separately and mixed after cooling. 10 mL of a solution of 10 g.L -1 AsIII (As 2 O 3 ), filtered at 0.22 µm, 7 were added to the culture medium. A concentrated solution of 50 g/L yeast extract was prepared, filtered at 0.22 µm and then stored at 4°C.

aioA gene expression with 75 mg.L -1 AsIII

The aim of the first experiment was to determine the influence of a complex organic substrate, yeast extract, on the expression of the aioA gene during the growth of the bacteria, in the presence of 1 mM AsIII, a concentration that provides enough energy for the growth of "Thiomonas arsenivorans" in batch conditions [5].

"T. arsenivorans" inoculum was cultivated in 20 mL of CAsO1 medium (pH 6.0) containing 75 mg.L -1 AsIII without organic substrate, in a cottoned 40 mL glass bottle. The inoculum of H. arsenicoxydans was prepared in CAsO1 medium (pH 7.2) containing 75 mg.L -1 AsIII and supplemented with 1 g.L -1 yeast extract. Two CAsO1 culture media containing, respectively, 0.2 and 1 g.L -1 yeast extract were inoculated, at pH 7.2 for H. arsenicoxydans and pH 6.0 for "T. arsenivorans". All flasks were incubated aerobically at 25°C under stirring. At t=0 and then twice per day, bacterial growth was monitored by enumerating bacterial cells with a Thoma cell under an optical microscope and by absorbance measurements at 620 nm. 1.5 mL of each culture were sampled in a 2 mL sterile microtube and centrifuged for 10 min at 5,000 x g. Pellets were frozen in liquid nitrogen and stored at -80°C until RNA/DNA coextraction. In parallel, 2 mL samples were filtered at 0.22 microns in sterile flasks and kept at 4°C until further AsV analysis. Abiotic controls prepared in the same conditions showed that AsIII was not oxidized abiotically [START_REF] Lescure | Influence of organic matters on AsIII oxidation by the microflora of polluted soils[END_REF].

AsIII oxidation kinetics

The aim of the second experiment was to quantify the influence of simple and complex organic substrates on the AsIII-oxidation rate when concentrations of AsIII are so low that the kinetics must be carried out over a short time, i.e. less than one hour. For this purpose, a 8 standardized cell concentration was used, allowing easier comparison of the results obtained in the different conditions. Acetate was used for H. arsenicoxydans as it is the only simple organic substrate used as the sole source of carbon and energy by this strain [START_REF] Muller | Herminiimonas arsenicoxydans sp. nov., a metalloresistant bacterium[END_REF]. For "T.

arsenivorans", which does not use acetate, succinate was chosen, since it is a simple organic substrate used efficiently by this strain [5]. Yeast extract was chosen as a complex organic substrate used by both strains, allowing comparison of results with a common substrate. Each strain was sub-cultured (1% volume) into 100 mL of CAsO1 medium containing 2 mg.L -1 of AsIII at the appropriate pH (pH 6.0 for "T. arsenivorans", pH 7.2 for H. arsenicoxydans) and different concentrations of organic substrate: 0.05, 0.2 or 1 g.L -1 yeast extract for both strains; 0.05, 0.2 or 1 g.L -1 of succinate for "T. arsenivorans" or acetate for H. arsenicoxydans.

Incubations were carried out aerobically for 3 days at 25°C under static conditions, to avoid the formation of cell aggregates (oxygen was not limiting). For all of the sub-culture conditions (0.05, 0.2 and 1 g.L -1 of organic substrates), two conditions were compared in triplicate to determine AsIII oxidation kinetics: the liquid phase of the bacterial suspension that oxidized 2 mg/L AsIII was either deprived of organic substrate, or contained 0.2 g.L -1 of organic substrate.

Appropriate sub-culture volumes were sampled at the end of the growth phase in order to adjust the final cell densities in the suspensions to values close to 2 x 10 7 cells.mL -1 . After centrifugation (30 minutes at 8,500 x g), pellets were washed with 20 mL of arsenic-free CAsO1 medium. After a second centrifugation (30 minutes at 8,500 x g), pellets were resuspended in 130 mL of arsenic-free CAsO1 medium, with or without 0.2 g.L -1 of organic substrate. 20 mL of the resulting bacterial suspensions were distributed in 60 mL sterile cottoned flasks. At t=0, 400 µL of 100 mg.L -1 AsIII solution were added to the flasks to obtain a concentration of 2 mg.L -1 AsIII, mixed immediately and sampled for bacterial cells counting and for arsenic 9 speciation. The flasks were incubated at 25°C, under static conditions, and AsIII oxidation was monitored over 45 minutes, with 2.5 mL-sampling every 15 minutes. The samples were filtered at 0.2 µm in sterile microtubes immediately after sampling and were promptly diluted tenfold in sterile ultrapure water and stored at 4°C for subsequent As speciation. Blank experiments were prepared in the same conditions without cells. The specific AsIII-oxidation rate was calculated as the ratio of linear AsIII oxidation over 45 minutes and the cell concentration measured at the start of the experiment (this concentration did not significantly vary during the 45 minutes of the experiment).

Speciation and quantification of arsenic

AsIII and AsV were separated using resins (AG® 1-X8 Resin, Cat. # 140-1431, Biorad) as described in [START_REF] Battaglia-Brunet | A simple biogeochemical process removing arsenic from a mine drainage water[END_REF], and a fraction of the sample remaining in the tube was used for total As quantification. Quantification of As in tubes containing AsV, AsIII and total As was carried out by electrothermal atomic absorption spectrophotometry (Varian SpectrAA 220Z).

RNA and DNA co-extraction and cDNA synthesis

RNA and DNA co-extractions were performed on cell pellets stored at -80°C, using the NucleoSpin® RNA II kit (Macherey-Nagel) in accordance with the manufacturer's recommendations, including a separate DNA elution step with the NucleoSpin® DNA/RNA Buffer Set (Macherey-Nagel). The column was then placed on a new 1.5 mL collection tube and the manufacturer's protocol was continued with the digestion of DNA followed by RNA elution. An additional digestion step was needed to remove residual DNA from the RNA extract. To that end, extracted RNA was mixed with 7 µL of buffer and 1 µL of Turbo DNase (2 U; Ambion, ThermoScientific) and then incubated at 37°C for 10 min. Finally, a new RNA purification step was performed with the NucleoSpin® RNA II kit (Macherey-Nagel). The 10 RNA was finally eluted in 60 µL and assayed by BioPhotometer spectrophotometer (Eppendorf). The quality of extracted RNA was checked on 1% agarose gel (25 min at 100 V) and by calculation of the A 260 /A 230 and A 260 /A 280 ratios. They were stored in LoBind microtubes (Eppendorf), at -80°C until the reverse-transcription PCR (RT-PCR) step. The reaction mixture used for the RT-PCR step consisted of 1 µL iScript™ reverse transcriptase (BioRad), a variable volume of RNA extract (so as to obtain 50 ng per tube), 4 µL 5X ™ iScript reaction mix (BioRad), and ultrapure nuclease-free water (qsp 20 µL). RT-PCR consisted of 5 min at 25°C, 30 min at 46°C, 5 min at 85°C and hold at 15°C.

aioA gene and transcripts quantification by qPCR

The aioA gene involved in AsIII oxidation and its transcripts were quantified by quantitative real-time PCR (qPCR), respectively from DNAs and cDNAs obtained from cultures of "T.

arsenivorans" and H. arsenicoxydans. qPCR was carried out using m4-1F forward (GCCGGCGGGGGNTWYGARRAYA) and m2-1R reverse (GGAGTTGTAGGCGGGCCKRTTRTGDAT) primers, as designed in [START_REF] Quéméneur | Les processus biogéochimiques impliqués dans la mobilité de l'arsenic : recherche de bioindicateurs[END_REF] and applied in [START_REF] Fernandez-Rojo | Biological attenuation of arsenic and iron in a continuous flow bioreactor treating acid mine drainage (AMD)[END_REF]. The expected product size was 110 bp. Each primer was used at a concentration of 0.3 µM, with 100 ng of T4GP32 (MP Biomedicals), in 1X IQ SYBR Green Supermix (BioRad) and a final volume of 20 µL. The program was run on a CFX Connect (BioRad) and consisted of an initial denaturation at 95°C for 3 min, followed by 50 cycles of 95°C for 10 s, 54°C for 20 s, 72°C for 10 s, and a data acquisition step at 80°C for 30 s. At the end, a melting curve analysis was performed by measurement of the SYBR Green I signal intensities during a 0.5°C temperature increment every 10 s, from 65°C to 95°C. Negative template controls confirmed the absence of contaminant DNA. Six-point serial decimal dilution of a linearized plasmid carrying the H. arsenicoxidans aioA gene was used to generate a linear calibration curve of threshold cycle versus a number of gene copies ranging from 10 1 to 10 6 .

11

All measurements were run in duplicates. Results were expressed as ratios of aioA transcripts relative to the aioA gene (aioA mRNA/DNA ratio).

Statistical analysis

Statistical analysis were performed with XLSTAT 2019: normality of the data was first verified using the tests of Shapiro-Wilk, Anderson-Darling, Lilliefors and Jarque-Bera. Then, significance of differences between results obtained at different substrate concentrations was evaluated using an Anova parametric test. Differences between modalities were analysed with a confidence interval of 95% using the methods of Tukey and Dunnett.

Results

Expression of aioA gene during bacterial growth in the presence of 75 mg.L -1 AsIII

Arsenite oxidation activity in batch conditions was monitored together with the detection and quantification of aioA gene expression and growth of the pure strains. Fig. 1A. shows that expression of the aioA gene by "T. arsenivorans" was mainly detectable during early growth (Fig. 1C). Maximum aioA gene expression was measured at 24 h, corresponding to about 60 mg.L -1 , i.e. 80% of oxidized AsIII (Fig. 1E), and tended to be higher in the presence of 0.2 g.L -1 than 1 g.L -1 of yeast extract (average aioA mRNA/DNA ratios were, respectively, 0.032 and 0.0015 in the presence of 0.2 g.L -1 and 1 g.L -1 yeast extract). However, due to a large variability between replicates, the difference observed between ratios obtained at 0.2 and 1 g/L yeast extract was not statistically significant. Expression of the aioA gene for H. arsenicoxydans was detectable throughout the duration of the experiment (Fig. 1B). As for "T. arsenivorans", maximum expression was detected in the early growth (Fig. 1D) and AsIII oxidation phase (t=5 h), and corresponds to 20-30 mg.L -1 (26-40%) of oxidized AsIII (Fig. 12 1F). Here, in the maximum aioA gene expression phase, expression was significantly greater in the presence of 0.2 g.L -1 than 1 g.L -1 of yeast extract (at t=5h, average aioA mRNA/DNA ratios were 1.6 x 10 -3 and 3.3 x 10 -4 in the presence of 0.2 g.L -1 and 1 g.L -1 of yeast extract, respectively).

For T. arsenivorans, AsV concentrations were always similar whatever the concentrations of yeast extract (Fig 1E ), whereas for H. arsenicoxydans, AsV concentration was slightly higher with 1 g.L -1 yeast extract at t=5h (Fig. 1F). No significant AsV oxidation was observed in sterile media (Fig. 1G). For both strains, bacterial growth was higher at 1 g.L -1 than at 0.2 g.L - 1 of yeast extract (Fig. 1C and1D). Consequently, the lower the yeast extract concentration, the higher the specific AsIII oxidation.

Oxidation kinetics with 2 mg.L -1 AsIII

Cells at the end of the growth phase, that grew at different concentrations of a simple organic substrate or yeast extract oxidized AsIII in a minimum medium containing 2 mg.L -1 of AsIII and no organic substrate (Fig. 2) (cell growth was negligible throughout the 45 min duration of the AsIII-oxidation kinetics). In the blank experiments without cells, AsV concentration remained lower than the quantification limit. Examination of the kinetics shows that oxidation of AsIII was more efficient with cells that grew on yeast extract (Fig. 2B and2D) than on simple substrates (Fig. 2A and2C), and more efficient with "T. arsenivorans" (Fig. 2A and2B) than with H. arsenicoxydans (Fig. 2C and2D). H. arsenicoxydans did not grow well on acetate, thus the rates obtained with this strain and a simple substrate were very low (Fig. 2C).

The results expressed as specific oxidation rates (Fig. 3) confirm these observations: for both strains, a significant decrease of specific AsIII oxidation rates was observed when yeast extract concentration in the growth medium increased (Fig. 3A). The same trend was 13 observed for "T. arsenivorans" grown on succinate as a simple organic substrate (Fig. 3B). H. arsenicoxydans did not grow well in the presence of acetate, although the trend is less clear.

However, for this strain, the specific AsIII oxidation rate was lower at 1 g.L -1 (5.6.10 -11 µg/cell/min) than at 0.05 g.L -1 of acetate (2.10 -10 µg/cell/min).

These specific rates were compared with those obtained when AsIII-oxidation kinetics were determined in the presence of organic substrates (Fig. 4 and Table 1). A negative effect of the addition of yeast extract was observed systematically for both strains, whatever the preculture condition: rates decreased by 13.6% to 39.1% for "T. arsenivorans" and by 10% to 25.9% for H. arsenicoxydans. In contrast, no effect on AsIII-oxidation rates was detected with simple organic substrates (acetate or succinate).

Discussion

A negative effect of organic substrates on bacterial AsIII-oxidation activity was observed with two pure strains presenting contrasting metabolisms, one able to use AsIII as an energy source ("T. arsenivorans"), the other not (H. arsenicoxidans). This study has shown, for the first time, the negative effect of a complex organic substrate, yeast extract, on the expression of the aioA gene encoding the catalytic subunit of the AsIII-oxidase in the Aio system. So far, the main factor studied influencing the expression of the aioA gene was the presence and concentration of arsenic III or V. Expression of the aioA gene is induced by the presence of As in most AsIII-oxidizing bacteria [START_REF] Slyemi | How prokaryotes deal with arsenic[END_REF] including several strains belonging to the Thiomonas genus [START_REF] Hovasse | Spatio-Temporal Detection of the Thiomonas Population and the Thiomonas Arsenite Oxidase Involved in Natural Arsenite Attenuation Processes in the Carnoulès Acid Mine Drainage[END_REF]. However, in some strains, aioA gene expression is also observed in the absence of arsenic: Thiomonas sp. 3AS [START_REF] Duquesne | Arsenite oxidation by a chemoautotrophic moderately acidophilic Thiomonas sp. : from the strain isolation to the gene study[END_REF], Ralstonia sp. 22 [START_REF] Lieutaud | Arsenite oxidase from Ralstonia sp. 22: characterization of the enzyme and its interaction with soluble cytochromes[END_REF] and Agrobacterium tumefaciens 5A strain, for which AsIII oxidation seems regulated by quorum-sensing [START_REF] Kashyap | Complex regulation of arsenite oxidation in Agrobacterium tumefaciens[END_REF], and for some 14 strains of Pseudomonas [START_REF] Poirel | Arsenite-induced changes in abundance and expression of arsenite transporter and arsenite oxidase genes of a soil microbial community[END_REF][START_REF] Koechler | Constitutive arsenite oxidase expression detected in arsenic-hypertolerant Pseudomonas xanthomarina S11[END_REF]. In H. arsenicoxydans, the expression of genes in the aio operon is related to the presence of AsIII and to the expression of genes involved in motility [START_REF] Koechler | Multiple controls affect arsenite oxidase gene expression in Herminiimonas arsenicoxydans[END_REF]. Our results are in accordance with those of Nandre et al. [START_REF] Nandre | Enhanced Detoxification of Arsenic Under Carbon Starvation: A New Insight into Microbial Arsenic Physiology[END_REF], who observed a higher expression of aioA genes under acetate starvation condition in seven strains belonging to different genera, for one single time of incubation (8 h), in presence of 1.33 mM of As. Here, the phenomenon was observed with a complex organic substrate (yeast extract) and correlated with early growth phase.

The specific AsIII-oxidation rates obtained as part of this study are of the same order of magnitude (Table 2) as the V max of AsIII oxidation determined for Variovorax sp. 24 [START_REF] Janda | 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls[END_REF] and Agrobacterium albertimagni AOL15 [START_REF] Salmassi | Oxidation of arsenite by Agrobacterium albertimagni AOL15, sp. Nov., isolated from hot creek California[END_REF].

The specific oxidation rate was lower when cells had been grown with a high substrate concentration. Several authors have observed that simple or complex organic substrates influence the activity of AsIII-oxidizing bacteria. Santini et al. [START_REF] Santini | A new chemolithoautotrophic arseniteoxidizing bacterium isolated from a gold mine: phylogenetic, physiological, preliminary biochemical studies[END_REF] reported that the AsIIIoxidizing strain NT-26, able to use AsIII for energy, showed strong AsIII-oxidase activity when it was grown without any organic substrate, the addition of yeast extract resulting in a decrease in AsIII-oxidizing activity. Challan-Belval et al. [START_REF] Challan-Belval | Enhancing pozzolana colonization by As(III)-oxidizing bacteria for bioremediation purposes[END_REF] observed that AsIII oxidation by a bacterial consortium containing "T. arsenivorans", grown as biofilm in microplate experiments with pozzolana, was drastically decreased in the presence of yeast extract, dropping from 100% to 34%. This result was explained by the supply of nutrients present in yeast extract, including electron donors other than AsIII. Bachate et al. [START_REF] Bachate | Oxidation of arsenite by two betaproteobacteria isolated from soil[END_REF] studied the influence of various parameters (temperature, pH and growth medium) on AsIII-oxidation rate by bacterial cells washed and re-suspended in 75 mg.L -1 AsIII-containing medium. They followed AsIII oxidation by these cells for 1 hour. The strains were heterotrophic bacteria, phylogenetically related to the genera Bordetella and Achromobacter. These authors observed that the growth conditions have an effect on AsIII-oxidation rate: the latter was maximal when the strains were cultured with only 0.4 g.L -1 yeast extract, decreased by 25 to 50% with 1 g.L -1 yeast extract, and by 85% with 0.4 g.L -1 of yeast extract and 20 mM (1.18 g.L -1 ) of acetate. They also observed a decrease in the enzymatic AsIII-oxidase activity of both strains with the increase of total organic substrate concentration in the growth medium. The present results are consistent with those of Bachate et al. [START_REF] Bachate | Oxidation of arsenite by two betaproteobacteria isolated from soil[END_REF], although the studied strains belong to different genera and come from very different environments: "T. arsenivorans" and H.

arsenicoxydans were isolated from a mining site and a wastewater treatment plant respectively, whereas the origin of strains of Bachate and al. [START_REF] Bachate | Oxidation of arsenite by two betaproteobacteria isolated from soil[END_REF] was a garden soil.

Here, bacterial specific AsIII-oxidation rate was adversely affected for both strains by the presence of simple or complex organic substrates in growth medium. This phenomenon was observed both at relatively high AsIII concentrations (75 mg.L -1 ) and at the lower concentrations of arsenic (2 mg.L -1 ) commonly detected in groundwater or soil pore water on polluted sites. This negative effect on AsIII oxidation was observed with a complex organic substrate (yeast extract), as well as with at less one simple organic substrate that do not provide nitrogen, i.e. succinate, suggesting that the observed phenomenon is not linked to nitrogen metabolism but rather to energy or carbon source. Thus for both "T. arsenivorans", a mixotrophic strain using AsIII as energy source, and H. arsenicoxydans, a heterotrophic strain, arsenic oxidation and metabolism of organic substrates seem to be linked.

Several hypotheses may explain the negative effect of organic substrates on aioA gene expression and the bacterial specific AsIII-oxidation rate. Nandre et al. [START_REF] Nandre | Enhanced Detoxification of Arsenic Under Carbon Starvation: A New Insight into Microbial Arsenic Physiology[END_REF] hypothesized cross protection in the carbon starvation stress wherein nutrient deprived cells show higher resistance against other stresses. However, this phenomenon could hardly explain results obtained with bacteria such as "T. arsenivorans" usually growing in mineral or oligotrophic media. In bacteria using AsIII as an energy source, such as NT-26 or "T. arsenivorans", the phenomenon could be related to competition between AsIII and organic substrates as an 16 energy source: in the presence of high energy-providing organic molecules, cells do not have to acquire energy from the AsIII oxidation reaction, and therefore express the aioA gene more weakly. In bacteria unable to gain energy from AsIII oxidation (like H. arsenicoxydans) and in which the ability to oxidize AsIII can be a detoxification mechanism [START_REF] Santini | A new chemolithoautotrophic arseniteoxidizing bacterium isolated from a gold mine: phylogenetic, physiological, preliminary biochemical studies[END_REF], the presence of a high concentration of organic substrate may have a protective effect against AsIII.

Another hypothesis would be linked to complexation reactions that may occur between As and some compounds present in complex organic substrates, and thus reduce AsIII toxicity and/or bioavailability. Here, the results of experiments at 2 mg.L -1 As suggest that the complex organic substrate (i.e. yeast extract) has a negative effect on AsIII oxidation, considering the comparison of kinetics with cells in suspension in solution with or without yeast extract. This phenomenon may be related to the complexation of AsIII with certain components of yeast extract such as thiol groups of amino acids that would make As less bioavailable. These complexation reactions would not occur with simple organic substrates.

Direct binding of AsIII to cysteine residues of proteins has been observed [START_REF] Kitchin | The role of protein binding of trivalent arsenicals in arsenic carcinogenesis and toxicity[END_REF]. Another possible explanation of the negative effect of simple or complex organic substrates on AsIII oxidation may be that the availability of energy sources would favor the AsV-reducing Ars arsenic resistance system [START_REF] Silver | Genes and enzymes involved in bacterial oxidation and reduction of inorganic arsenic[END_REF] because this resistance process consumes energy. Regarding the Ars resistance system, most studies have focused on arsB and ACR3 genes carriers [START_REF] Kashyap | Complex regulation of arsenite oxidation in Agrobacterium tumefaciens[END_REF][START_REF] Achour | Diversity of arsenite transporter genes from arsenicresistant soil bacteria[END_REF][START_REF] Achour-Rokbani | Characterization of the ars gene cluster from extremely arsenic-resistant Microbacterium sp. strain A33[END_REF] whose expression was shown to be controlled by the concentration of arsenic and phosphate. Further studies should focus on determination of the effect of organic substrates on AsIII complexation, and on the expression of the arsC gene encoding an AsV reductase in the Ars resistance system, which, in parallel with the Aio system, could influence the balance of speciation of inorganic arsenic in aerated or microaerophilic environments.

23

[46] Das S, Jean JS, Kar S, Chou ML, Chen CY. Screening of plant growth-promoting traits in arsenic-resistant bacteria isolated from agricultural soil and their potential implication for arsenic bioremediation. J Haz Mat 2014;272:112-120.

[47] Bahar MM, Megharaj M, Naidu R. arsenicoxydans (F) during this experiment, and in sterile culture media incubated in the same condition (G). Significance of differences between modalities 0.2 and 1 g.L -1 yeast extract was evaluated using an Anova parametric test (details in material and methods section).

Figure 1 .

 1 Figure 1. Expression of aioA gene represented as the aioA mRNA/DNA ratio during growth of T. arsenivorans (A) and H. arsenicoxydans (B) in the presence of 75 mg.L -1 AsIII. ;

Figure 2 .

 2 Figure 2. Kinetics of AsIII oxidation in minimum medium containing 2 mg/L AsIII and no

17

The authors declare that there is no conflict of interest regarding the publication of this article.

Aknowledgements

This work was supported by the French Environment and Energy Management Agency (ADEME), BRGM (convention , and the Labex Voltaire (ANR-10-LABX-100-01). We are grateful to the laboratory of Molecular Genetics, Genomics, Microbiology (GMGM) of Louis Pasteur University (Strasbourg, France) for providing the H. arsenicoxydans strain.

Conflict of interest

Significance of differences between modalities was evaluated using an Anova parametric test (details in material and methods section). Table 1. Influence of the presence of 0.2 g.L -1 organic substrate on specific AsIII oxidation rate during the kinetics: variation of AsIII oxidation rates between 0 and 0.2 g.L -1 organic substrates conditions, with pre-cultures grown under different conditions. Significance of variation was tested by ANOVA parametric test.

Strain

Pre-culture conditions Variation (%) of AsIII oxidation rates between 0 and 0.


Pritam Das

Simona Salerno

Jean-Christophe Remigy

Jean-François Lahitte

Patrice Bacchin

Loredana De Bartolo

Double porous poly (Ɛ-caprolactone)/chitosan membrane scaffolds as niches for human mesenchymal stem cells

Keywords:

In this paper, we developed membrane scaffolds to mimic the biochemical and biophysical properties of human mesenchymal stem cell (hMSC) niches to help direct self-renewal and proliferation providing to cells all necessary chemical, mechanical and topographical cues. The strategy was to create three-dimensional membrane scaffolds with double porosity, able to promote the mass transfer of nutrients and to entrap cells. We developed poly (Ɛ-caprolactone) (PCL)/chitosan (CHT) blend membranes consisting of double porous morphology: (i) surface macrovoids (big pores) which could be easily accessible for hMSCs invasion and proliferation; (ii) interconnected microporous network to transfer essential nutrients, oxygen, growth factors between the macrovoids and throughout the scaffolds. We varied the mean macrovoid size, effective surface area and surface morphology by varying the PCL/CHT blend composition (100/0, 90/10, 80/20, 70/30). Membranes exhibited macrovoids connected with each other through a microporous network; macrovoids size increased by increasing the CHT wt%. Cells adhered on the surfaces of PCL/CHT 100/0 and PCL/CHT 90/10 membranes, that are characterized by a high effective surface area and small macrovoids while PCL/CHT 80/20 and PCL/CHT 70/30 membranes with large macrovoids and low effective surface area entrapped cells inside macrovoids.

The scaffolds were able to create a permissive environment for hMSC adhesion and invasion promoting viability and metabolism, which are important for the maintenance of cell integrity. We found a relationship between hMSCs proliferation and oxygen uptake rate with surface mean macrovoid size and effective surface area. The macrovoids enabled the cell invasion into the membrane and the microporosity ensured an adequate diffusive mass transfer of nutrients and metabolites, which are essential for the long-term maintenance of cell viability and functions.

Introduction

An important challenge in tissue engineering and regenerative medicine is the design of a biomaterial able to provide all necessary physical, chemical and mechanical cues that promote cell colonization and tissue regeneration. Design approach for biomaterials are driven to the interest to reproduce the natural niche of cells that implies cell-cell and cell-extracellular matrix (ECM) contacts in a 3-D environment which ensure both the maintenance of cell polarity and cell-cell biochemical communication. In this context, a great interest is focused on the development of materials able to boost the proliferation and maintenance of mesenchymal stem cells (MSCs) secreting a broad spectrum of macromolecules [START_REF] Salerno | Gas permeable membrane bioreactor for the co-culture of human skin derived mesenchymal stem cells with hepatocytes and endothelial cells[END_REF][START_REF] Piscioneri | Neuroprotective effect of human mesenchymal stem cells in a compartmentalized neuronal membrane system[END_REF] which represent a promising source for cell therapy and regenerative medicine. It has been reported that the hMSCs viability and functions highly depend on environmental cues such as oxygen and nutrient availability, pore size, scaffold mechanical property, surface morphology and more notably the administration of soluble factors [START_REF] Di Luca | Gradients in pore size enhance the osteogenic differentiation of human mesenchymal stromal cells in three-dimensional scaffolds[END_REF][START_REF] Kasten | Porosity and pore size of β-tricalcium phosphate scaffold can influence protein production and osteogenic differentiation of human mesenchymal stem cells: an in vitro and in vivo study[END_REF]. Moreover, the hMSCs incorporation in a threedimensional bulk environment, that ensure the mass transfer of nutrients and growth factors to and from the entrapped cells, represents a further challenging strategy in next advanced co-culture systems with other target cells. Microporous membranes with suitable topographical, mechanical, physico-chemical and permeability properties can act as an artificial stem cell niche providing a bio-instructive extracellular environment characterized by micro-and nano-architecture [START_REF] Morelli | Recent strategies combining biomaterials and stem cells for bone, liver and skin regeneration[END_REF][START_REF] Salerno | Advanced membrane systems for tissue engineering[END_REF]. Membranes enable to provide an adequate structure in terms of porosity and interconnected pores, which is important for the selective mass transfer of nutrients and metabolites, and on the other hand favour cell ⁎ Corresponding author.

E-mail addresses: bacchin@chimie.ups-tlse.fr (P. Bacchin), l.debartolo@itm.cnr.it, loredana.debartolo@cnr.it (L. De Bartolo).

https://doi.org/10.1016/j.colsurfb.2019.110493 casting knife in order to achieve a thickness of 250 μm, at room temperature. Then the track-etched membrane, a polyethylene terephthalate (PET) isoporous membrane (Sterlitech, USA) with pore diameter 10 μm, was rinsed by the solvent, slowly wiped to remove excess solvent, and gently applied on the casted polymer solution (Fig. S1).

The glass plate was then slowly immersed inside the non-solvent bath containing NaOH aqueous solution at room temperature. Just after immersion, solvent exchange started by forming a flat sheet membrane due to de-mixing of the polymer solution. A homogeneous solvent-exchange, on the casted solution not covered by the track-etched membrane, led to a single and closed porous structure. On the other hand, two different solvent-exchange rates took place in polymer solution covered by the track-etched membrane, which led to the double porous morphology. The macrovoids were formed where the non-solvent could manage a direct access inside the casted solution through the pores of the track-etched membrane and an interconnected microporous network was formed where the non-solvent was not able to make a direct entry.

CHT was dissolved in the solvent at 55 °C for 12 h. PCL was successively added when the temperature was below 35 °C. Within 2 h a clear, viscous and a faint yellow solution was obtained. After mixing, the polymeric solutions were kept in stand for 10-20 min and then cast. Due to high viscosity of CHT in the solvent, it was quite difficult to make a homogeneous solution by mechanical stirrer condition with PCL/CHT ratio beyond 70/30, which could cause phase segregation between the two polymers. Double porous membrane formation with pure CHT was also not possible as dissolving CHT more than 4 wt% was difficult by mechanical stirring condition and that wt % is not enough to make a practically workable membrane due to lack of good entanglement between the polymer chain.

After formation of the membrane the track-etched membrane was removed gently and the newly formed membrane was washed several times in pure water. Then, the membranes were stored in ultrapure water at 4 °C temperature. The surface where the macrovoids were open towards the non-solvent are denoted as top surface and the other surface, which was facing the glass plate, denoted as the bottom surface.

Membrane characterization

After the preparation, membranes were characterised in order to determine their morphological, physico-chemical, permeability and mechanical property.

The surface morphology and cross section were analysed by scanning electron microscopy (SEM, Quanta 200F, FEI, USA). Diameter of the macrovoids and micropore size distribution were evaluated by ImageJ software on the SEM images by considering at least 10 micrographs of each membrane with minimum average area 10 4 μm 2 . The mean diameter of the passing pores was determined by Capillary Flow Porometer (CFP 1500 AEXL, Porous Materials Inc., PMI, Ithaca, New York, USA).

The membrane permeability was evaluated in an Amicon cell by pure water flux (J solvent ) measurements in the absence of solutes and at transmembrane pressures (ΔP™) ranging from 20 to 60 kPa. The hydraulic permeance Lp was measured assuming a linear correlation between the water flux (J solvent ) and the convective driving force, according the equation:

Lp = (J solvent /ΔP™) Δc = 0 . (1)
The mechanical properties were determined by Zwich/Roell Z2.5 tensile testing machine (Germany), by applying a pre-load of 0.05 MPa at constant 4 mm/min elongation rate [START_REF] Salerno | Polymeric membranes modulate human keratinocyte differentiation in specific epidermal layers[END_REF]. Tensile strength (N/mm 2 ), Young's modulus E (N/mm 2 ) and elongation at break ε (%) were determined at room temperature on ten different samples (1 × 5 cm) from each batch, considering the different cross-section thickness, in dry and attachment on the surface and invasion inside the bulk, increasing the effective area for cell accommodation [START_REF] Ahmed | 3D liver membrane system by co-culturing human hepatocytes, sinusoidal endothelial and stellate cells[END_REF][START_REF] Drioli | Membrane bioreactor for cell tissues and organoids[END_REF]. However, this balance between the optimal pore size and effective cell adhesion area is often compromised in the case of small pores suitable for the transfer of nutrients, growth factors and waste products that limit the cell proliferation to the surface. Conversely, in the case of big pores, it implies an increase of area for cell invasion and migration within the bulk limiting the selective transport of molecules and the initial interaction with the surface, which is crucial to mediate all subsequent events such as proliferation and differentiation [START_REF] O'brien | The effect of pore size on cell adhesion in collagen-GAG scaffolds[END_REF][START_REF] Murphy | The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering[END_REF]. Big pores are also responsible to reduce the mechanical strength and can drive a faster degradation in vivo before finishing the tissue construct. To overcome these drawbacks, membranes with double porosity can be a further step forward where the macrovoids (big pores) are responsible for the invasion and proliferation of the cells, and the transport of nutrients and growth factors can be accomplished by interconnected micropores, resulting a higher effective surface area than a single big porous surface. Previously, we developed a polysulfone (PSU) membrane with double porosity level for cell culture that supported only partially the cell adhesion and migration owing to the hydrophobic and non-degradable character of the PSU [START_REF] Dufresne | Human hepatic cell behavior on polysulfone membrane with double porosity level[END_REF]. Based on this previous concept here we developed more biocompatible and biodegradable membranes with double porosity through the modified liquid induced phase inversion process, by applying commercial track-etched membrane. The idea was to create new blended double porous membranes that can combine the features of chitosan (CHT) (e.g., bioactive functionality, hydrophilicity, degradation rate, chemical structure resemblance of the native tissue ECM) and polycaprolactone (PCL) (e.g., good mechanical properties, easy processing ability) [START_REF] Salerno | Dermalepidermal membrane systems by using human keratinocytes and mesenchymal stem cells isolated from dermis[END_REF][START_REF] Salerno | Polymeric membranes modulate human keratinocyte differentiation in specific epidermal layers[END_REF][START_REF] Neves | Chitosan/poly(ε-caprolactone) blend scaffolds for cartilage repair[END_REF], reducing the limitations of a single component and providing a greater level of control over the overall material properties for cell guidance and biocompatibility [START_REF] Mitragotri | Physical approaches to biomaterial design[END_REF][START_REF] Meyers | Structural biological materials: critical mechanics-materials connections[END_REF]. Interestingly, the bioactive functionality, biocompatibility, degradation and mechanical properties of the polymeric PCL-CHT blends have disclosed promising results in comparison to that on the separate culturing substratum [START_REF] Salerno | Dermalepidermal membrane systems by using human keratinocytes and mesenchymal stem cells isolated from dermis[END_REF][START_REF] Salerno | Polymeric membranes modulate human keratinocyte differentiation in specific epidermal layers[END_REF][START_REF] Neves | Chitosan/poly(ε-caprolactone) blend scaffolds for cartilage repair[END_REF]. Membranes must have an appropriate architecture to host cells at high density and to ensure an adequate transport of nutrients and metabolites [START_REF] Morelli | Human hepatocyte functions in a galactosylated membrane bioreactor[END_REF][START_REF] Bartolo | Human lymphocyte PEEK-WC hollow fiber membrane bioreactor[END_REF]. To achieve this goal, membranes should have macrovoids to accommodate cells, and a microporosity for diffusion of nutrients. These requirements are essential for cell nutrition, proliferation and invasion inside the bulk for vascularization and formation of new tissues. For the first time we report here the design and application of a 3D double porous membrane scaffold of PCL-CHT prepared by modified liquid induced phase inversion technique for the culture of hMSCs. The scaffolds consist of macrovoids (22-46 μm) which are open towards the surface for cell proliferation and invasion, and interconnected micropores to transfer essential nutrients, growth factors and cell catabolites. Membranes were characterised in order to evaluate the structural, mechanical and physico-chemical properties before their use in contact with cells. In particular, we explored the capability of the double porous membranes to create a physical and chemical niche for hMSCs through investigation of cell adhesion, proliferation and invasion as well as the metabolic oxygen requirements.

Materials and methods

Membrane fabrication with 3D double porosity

The membranes were prepared using a liquid induced modified phase inversion technique by diffusion between solvent as formic acid (FA)/acetic acid (AA) mixture and non-solvent NaOH solution. In particular, poly (Ɛ-caprolactone) (MW 80 KDa, CAPA™ 6800, Perstorp Holding AB, Sweden) and CHT (MW 190-310 KDa, 75-85 % degree of de-acetylation, Sigma-Aldrich), with ratio 100/0 (15 wt%), 90/10 (14 wt%), 80/20 (14 wt%) and 70/30 (10 wt%) (w/w %) were dissolved in FA/AA (w/w %) mixture until complete dissolution. The polymer solutions were individually cast on a glass plate by a handle-

= × - W W W W 100 loss i f i (2)

Cell culture

Human bone marrow mesenchymal stem cells (hMSCs) StemPro™ BM (Thermo Fisher Scientific) were cultured on sterilised double porous membranes with culture medium constituted by MesenPRO RS™ (Thermo Fisher Scientific) supplemented with 2 mM glutamine, 1% gentamycin sulphate/amphotericin-B, and 2% serum. hMSCs were seeded on the membrane scaffolds at 3.5*10 3 cell/cm 2 density, and incubated at 37 °C in a 5% CO 2 /20% O 2 atmosphere (v/v) with 95% relative humidity. Culture medium was changed every 48 h, and cells maintained up to 21 days.

Effective surface area (ESA%) for cell viability

We determined the effective surface area (ESA%) on the SEM images of the scaffolds by imageJ analysis. We calculated the total area of the macrovoids on a unit surface and subtract it from the total unit surface area as follows:

= ⎛ ⎝ ⎜ ⎜ ⎜ ⎜ - ⎞ ⎠ ⎟ ⎟ ⎟ ⎟ ×

Effective surface area ESA Total unit surface area

Total area of the macrovoids on that surface Total unit surface area

( %) 100 (3)

Cell morphology

Cell morphology was investigated after 7 and 21 days of culture on double porous membrane scaffolds by Scanning Electron Microscopy and Confocal Laser Scanning Microscopy.

hMSCs were examined by SEM (SEM, Quanta 200F, FEI, USA) after proper fixation and dehydration, as previously described [START_REF] Salerno | Gas permeable membrane bioreactor for the co-culture of human skin derived mesenchymal stem cells with hepatocytes and endothelial cells[END_REF]. Samples were gently washed with PBS buffer, and then incubated for 30 min in 3% glutaraldehyde and 1% formaldehyde mixture. Successively, they were fixed for 30 min in 1% OsO 4, and progressively dehydrated in ethanol solutions.

For Confocal Laser Scanning Microscopy (CLSM, Fluoview FV300, Olympus Italia) analysis hMSCs were immunostained for specific markers [START_REF] Salerno | Dermalepidermal membrane systems by using human keratinocytes and mesenchymal stem cells isolated from dermis[END_REF]. In order to visualize the cell distribution on the membranes, the cytoskeleton protein vimentin and cell surface antigen CD90 were stained. Vimentin was stained by using a rabbit polyclonal anti-human vimentin (Santa Cruz Biotechnology, Santa Cruz, CA), and a Cy™2conjugated AffiniPure donkey anti-rabbit IgG (Jackson Im-munoResearch Europe Ltd., Cambridge, UK); CD90 was marked by using a mouse monoclonal anti-human CD90 (eBioscience, San Diego, CA), and a Cy™3-conjugated AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch Europe Ltd., Cambridge, UK). Primary antibodies were incubated overnight at 4 °C, the secondary ones for 2 h at room temperature. Counterstaining of nuclei was performed with 0.2 μg/ml of 4′,6-diamidin-2 phenyl indole (DAPI) (Molecular Probes Inc., Eugene, OR) for 30 min.

The presence of hMSCs inside the macrovoids of the double porous membranes and the change of cell morphology from the surface towards the bulk of the membrane scaffolds were monitored with scan depth determination by CLSM in the z-scan mode (step size: 0.5 μm) (Fluoview 5.0 software, Olympus Corporation). Quantitative measurements of cell invasion and cell morphology along the z-axis were calculated by image analysis of CLSM micrographs (Fluoview 5.0 software, Olympus Corporation). Cell invasion was reported in terms of percentage of cell distribution on the top surface and in the bulk, at a distance of 15 μm. The change in cell morphology was measured and reported as frequency of cell surface area, by analysing overlapped CLSM images from all the double porous membrane scaffolds.

Cell proliferation

Cell viability and proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay. After 7, 14 and 21 days of culture on the different double porous membranes, hMSCs were incubated in 5 mg/ml of MTT solution for 4 h at 37 °C. The yellow tetrazolium MTT salt was reduced by the mitochondrial dehydrogenase in living cells to purple formazan crystals. This precipitate was extracted by using 1 ml per sample of a lysis solution constituted of 10% sodium dodecyl sulphate, 0.6% acetic acid in DMSO, in mild stirring for 30 min at 37 °C. Then formazan product was quantified by spectrophotometry at 570 nm wavelength.

Oxygen uptake rate (OUR) measurements

The Oxygen uptake rate (OUR) of hMSCs cultured on the different double porous membrane scaffolds was continuously monitored up to 21 days by the pre-calibrated sensor dish reader (SDR; OxoDish®-DW, PreSens Precision Sensing GmbH), a high-throughput, 24-channel reader for non-invasive detection of O 2 in special multi-dishes. O 2 detecting sensors, placed at the bottom of each well, and containing a luminescent dye, were excited every 5 min by the SDR placed below the multi-dish. The luminescence lifetime was read out noninvasively through the transparent bottom, and the dissolved oxygen detected with a resolution of ± 0.4% O 2 at 20.9% O 2, and a response time < 30 s. Tests were done in the 24 well special multi-dishes in which samples with cells and samples without cells (as controls) were placed.

Statistical analysis

All tests were performed in triplicate and repeated three times. Data are reported as mean ± standard deviation. Statistical significance of collected data was determined according to ANOVA followed by Bonferroni t-test (p < 0.05).

Results

Membrane properties

The developed membrane scaffolds displayed different morphological, physico-chemical, mechanical, permeability and biodegradation properties (Table 1). SEM images revealed on the top surface macrovoids that are interconnected with a highly microporous spongy network (Fig. 1). Macrovoids with mean size of 13 ± 4 μm were found on PCL double porous membrane. The increase of CHT concentration in the polymeric blend increased gradually the mean diameter of macrovoids at the active surface to values of 15 ± 5 μm, 21 ± 11 μm and 35 ± 25 μm for PCL/CHT 90/10, PCL/CHT 80/20 and PCL/CHT 70/ 30, respectively (Table 1). The cross section of the membranes revealed inside the bulk macrovoids with bigger volume and mean diameters of wet conditions. Wet samples were analysed after an incubation of 6 h in PBS buffer. Real-time longitudinal deformation measurements were obtained and analysed by testXpert® testing software.

The membrane biodegradation was evaluated by enzymatic incubation in two different solutions of Lipase from Aspergillus oryzae (0.12 U/ml) and human Lysozyme (1300 U/ml), respectively, in PBS buffer and 0.5 mg/ml NaN 3 , to emulate the enzymatic activities of human serum [START_REF] Aragón | Polymeric electrospun scaffolds for bone morphogenetic protein 2 delivery in bone tissue engineering[END_REF]. Before the incubation, three different samples (2 × 1.5 cm) from each batch were dried in vacuum oven at 37 °C for 72 h and then their initial weight (W i ) precisely measured. Then the samples were immersed in 1 ml of enzymatic solutions that were freshly changed every seven days. At predetermined time intervals, the samples were washed with copious distilled water, dried in vacuum oven at 37 °C for 72 h, and their final weight (W f ) precisely measured. The weight loss index (W loss %) was determined by the following equation: 22 ± 3 μm, 34 ± 10 μm, 27 ± 13 μm and 46 ± 4 μm for PCL/CHT 100/0, PCL/CHT 90/10, PCL/CHT 80/20 and PCL/CHT 70/30, respectively. Interconnected micropores in the range of 1-5 μm were visible for all membranes and size distribution become wider with the increase of CHT concentration in the blended membranes (Fig. S2). Mean passing pore diameters of 0.02 μm, 0.07 μm, 0.05 μm and 1.55 μm were measured by capillary flow porometer for PCL/CHT 100/0, PCL/ CHT 90/10, PCL/CHT 80/20 and PCL/CHT 70/30 membranes, respectively. Consistently the surface porosity was also depending on the ratio of CHT reaching values of 56% for PCL/CHT 70/30 membranes. The observed steady-state hydraulic permeance of the membranes calculated as slope of the pure water flux measurements versus transmembrane pressure (ΔP™) straightline follows the same trends of pore size and porosity: membrane permeability increased with increasing pore size and porosity, as expected.

Depending upon the mean macrovoids size and their density on the surface area, we calculated the effective surface area (ESA%) to relate to the cell adhesion and proliferation. We found a decrease of ESA with increasing the percentage of CHT reaching values ranging from 88 ± 1% for PCL/CHT 100/0 to 69 ± 2% for PCL/CHT 70/30 membranes.

The mechanical properties of the membranes evaluated in dry and in wet conditions are reported in Table 1. PCL/CHT 100/0 membranes displayed stable elastic properties in both dry and wet conditions, as evidenced by the moderate tensile modulus E (143.6 ± 14.1 and 131.6 ± 2.2 N/mm 2 in dry and wet conditions, respectively) in combination with elongation at break higher than 100%. The increase of CHT wt% in the blended membranes caused a gradual decrease of all the mechanical properties, including the tensile modulus, with respect to the pure PCL ones, reaching low values on the membrane of PCL/ CHT 70/30, as a result of the gain of more fragile properties. Passing from the dry to the wet conditions, the tensile modulus and the ultimate tensile strength decreased, and the elongation at break increased (Table 1).

The biodegradable properties of the membranes were investigated by using separately lipase and lysozyme solutions at concentrations similar to human serum. The weight loss of all membranes increased with time as shown in Fig. 2. As expected, the degradation of membranes augmented with the decrease of PCL concentration giving rise the highest percentage of weight loss with the PCL/CHT 70/30 membranes. The membrane weight loss in lipase solution was quite fast and higher than that in lysozyme solution. After 17 days of incubation in lipase the blended PCL/CHT membranes were completely degraded. On the contrary, low percentage of weight loss was measured in lysozyme solution.

Adhesion and invasion of hMSCs in the double porous membranes

The capability of the developed membranes to promote the adhesion, invasion and proliferation of mesenchymal stem cells was evaluated in the culture time. Cells adhered over the membrane surfaces and appeared flattened with elongated filopodia as highlighted by SEM's images (Fig. 3). During the culture time they proliferated covering the membrane surfaces as shown in Fig. 3. In the case of PCL/CHT 100/0 and PCL/CHT 90/10 the hMSCs adhered on the surface and exhibited a spindle-like morphology. On the other two membranes (PCL/CHT 80/20 and PCL/CHT 70/30), which are characterised by large macrovoids at the surface (21 and 35 μm, respectively) and smaller effective surface area, cells beside adhere to the surface they were entrapped into the macrovoids and displayed a more spherical shape.

We investigated the expression of CD90 and vimentin as specific markers of undifferentiated status and proliferation/migration capability of MSCs, respectively. Cells on all investigated membranes were positive for the cytoskeleton protein vimentin (in green) and cell surface protein CD90 (in red) (Fig. 4). In agreements with the SEM observations, here we also found that cells attached to PCL/CHT 100/0 and PCL/CHT 90/10 surfaces exhibited a high degree of spreading. It is interesting to note that in the case of PCL/CHT 80/20 and PCL/CHT 70/ 30 membranes the most of cells were entrapped inside the macrovoids and acquired a globular morphology due to the limited area available for cell adhesion and organization. Since the spreading is characterized by a substantial flattening of the cells and an increase in overall surface area we evaluated the distribution of the cell surface area on the different membranes (Fig. 5). The frequency distribution of the cell surface area confirmed that on PCL/CHT 100/0 and 90/10 the most of cells achieved a very widely surface area (between 2400 and 5000 μm 2 ) on the contrary on PCL/CHT 80/20 and 70/30 a peak of surface area is around 60-100 μm 2 as an indication of unspread cells entrapped inside the macrovoids.

For a deeper understanding of the cell invasion phenomenon inside the scaffolds, we accrued the CLSM images in z-scanning mode from surface towards the bulk (Fig. 6). Quantitative image analysis established that 95 ± 1.8% and 97 ± 2.2% of cells adhered on the surface of PCL/CHT 100/0 and PCL/CHT 90/10 membranes, respectively, and few cells invaded the bulk. On the other hand, on PCL/CHT 80/20 and PCL/CHT 70/30 cells were distributed on both surface and bulk, acquiring spread and globular morphology, respectively. We calculated 16 ± 0.4% and 15 ± 3.1% of cells on PCL/CHT 80/20 and PCL/CHT 70/30 surfaces, respectively. For both membrane scaffolds 84 ± 0.4% and 85 ± 3.1% of cells were inside the bulk, at a distance of 15 μm from the top surface of PCL/CHT 80/20 and PCL/CHT 70/30 membranes. Moreover, the cell invasion in the latter two scaffolds increased

Cell proliferation and oxygen uptake rate of hMSCs in the double porous membranes

The cell experiment results demonstrated a remarkable effect of macrovoids size and surface morphology on cell proliferation and behaviour. Fig. 7a reports the quantitative cell viability by MTT assay after 7, 14 and 21 days. Here we found that cells grew on all membranes, especially on PCL/CHT 100/0 that showed the highest rate of cell viability after 14 days. The cell growth was slower on membranes with high CHT wt% that are characterised by greater mean macrovoid size and surface porosity. However, after 21 days all the membranes (except PCL/CHT 70/30) displayed almost similar cell viability. This result suggests that the scaffolds created a permissive microenvironment for hMSCs and after 21 days they reached confluence on the surface, even if differences in cell proliferation rates were observed as a function of macrovoids size and surface porosity.

Considering that oxygen deficiency is a serious problem for the cells and inadequate oxygenation induces a decrease of cell metabolism, we These results are in agreement with the proliferation data and SEM images that clearly indicate a very high density of the hMSCs on the surface of PCL/CHT 100/0 followed by PCL/CHT 90/10, without significant dead cell fragments.

Discussion

The design of bioactive membrane scaffolds mimicking the physiologic environment during tissue formation is an important challenge in biomaterials and tissue engineering research. Herein, we developed double porous polymeric membrane scaffolds that recapitulate the 3D microenvironment, able to ensure the long-term stem cell viability and metabolism. A distinguishing feature of the scaffolds is the combination of macrovoids that allow the cell entrapment with a small interconnected microporous network that ensures the selective transfer of oxygen and nutrients and the removal of cell catabolites and products. By changing the CHT wt%, not only the chemical properties of the scaffolds were varied, but also their permeability and structural properties like macrovoid size, effective surface area, surface porosity and surface regularity was also modified. Finally, all these properties had a direct and strong influence on the morpho-functional behaviour of hMSCs. The formation of the unique double porous morphology by modified non-solvent induced phase inversion is due to the track-etched membrane placement on the casted solution that produced two different solvent-exchange rates. The pores of the track-etched membrane restricted the non-solvent entry inside the casted polymer, leading to the formation of macrovoids [START_REF] Dufresne | Human hepatic cell behavior on polysulfone membrane with double porosity level[END_REF]. The addition and increase of CHT wt % enhanced the viscosity of the casted solution inducing consequently an increase of the macrovoids size in the bulk with respect to the opening ones on the surface. On the contrary, without the use of the commercial track-etched membrane, the phase inversion was quite homogeneous leading to a single porous morphology without macrovoids, followed by dense skin formation [START_REF] Dufresne | Human hepatic cell behavior on polysulfone membrane with double porosity level[END_REF][START_REF] Remigy | Morphological characterization of a polymeric microfiltration membrane by synchrotron radiation computed microtomography[END_REF].

The polymer composition influenced mechanical, physico-chemical and degradation properties as well as the surface morphology and macrovoid size of the developed membrane scaffolds. The mechanical behaviour of PCL was modified by introducing and increasing CHT wt %, that leads to an increase of the macrovoids size and the porosity, and consequently to a decrease of the polymers bulk density in unit volume. From these data an intrinsic correlation between hydraulic performance and structural properties of the membranes emerge. The larger mean pore size and porosity of the 70/30 PCL/CHT membrane is responsible for a hydraulic permeance almost 2 times higher than that measured for the 100/0 one.

The double porosity affected the tensile strength of all membranes [START_REF] Olubamiji | Modulating mechanical behaviour of 3D-printed cartilage-mimetic PCL scaffolds: influence of molecular weight and pore geometry[END_REF], and was responsible of the decrease of the E modulus even in the blended membrane scaffolds, despite the introduction of the stiffer polymer chitosan. Moreover, the decrease of the tensile strength and Young's modulus could be due to the thermodynamic miscibility of the two polymers that affects their interfacial adhesion in contact surface and the yield strength of the matrix [START_REF] Correlo | Properties of melt processed chitosan and aliphatic polyester blends[END_REF]. Additionally, in wet condition the mechanical properties dramatically change for double porous blended membranes owing to an incorporation of water molecules inside the bulk, through the macrovoids and among the polymeric chains. Water incorporation re-established new hydrogen bonds with the CHT polar functional groups, by disabling the previous polymer-polymer interaction to some extent and leading polymers to slide over another under stress. This phenomenon was previously observed also in CHT and PCL/CHT nanoporous membranes, and is responsible for reduced tensile strength and Young's modulus, and for increased elongation at break, in wet condition and increasing CHT wt% [START_REF] Salerno | Dermalepidermal membrane systems by using human keratinocytes and mesenchymal stem cells isolated from dermis[END_REF][START_REF] Salerno | Polymeric membranes modulate human keratinocyte differentiation in specific epidermal layers[END_REF].

The enzymatic degradation is a very important physiological phenomenon which ensures enhanced cell-cell connection followed by tissue formation with time inside a 3D biodegradable scaffold. Particularly, lipase and lysozyme with human serum concentration simulated the physiological conditions that would be responsible for enzymatic dissolution of the different double porous membrane scaffolds in vivo as artificial patch [START_REF] Azevedo | Understanding the enzymatic degradation of biodegradable polymers and strategies to control their degradation rate[END_REF]. Lipase mainly degrades PCL and polymeric chains that consists of lipid or ester-like chemical structure; lysozyme mainly degrades CHT consisting of N-acetyl/deacetyl glucosamine linkages via oxidative-reductive chain scission, with a degradation kinetic that is inversely related to the deacetylation degree of CHT [START_REF] Anitha | Chitin and chitosan in selected biomedical applications[END_REF]. The slower degradation rate in lysozyme is mainly due to the different degradation mechanism and the lower CHT wt% in all blends [START_REF] Honma | Poly(ε-caprolactone)/chitin and poly(εcaprolactone)/chitosan blend films with compositional gradients: fabrication and their biodegradability[END_REF]. Nevertheless, the overall enzymatic degradation phenomenon was further governed by the easy accessibility of the enzymatic solution throughout the bulk, which could be beneficial for a scaffold with fast cell growth, needing the scaffold to degrade faster while being replaced by cell-secreted ECM [START_REF] Mondschein | Polymer structure-property requirements for stereolithographic 3D printing of soft tissue engineering scaffolds[END_REF]. In particular, the lower lipase degradation of PCL/CHT 100/0 could be due to a higher difficulty of water-soluble Decreasing the concentration of PCL wt% the degradation increases as CHT chains decrease the crystallinity of PCL chains and also increase the hydrophilicity of the overall blends, creating more space for the enzymatic solution to invade inside the PCL chains [START_REF] He | Enzyme-catalyzed polymerization and degradation of copolymers prepared from ε-caprolactone and poly(ethylene glycol)[END_REF]. Moreover, increasing the macrovoids size and porosity, the enzyme can have a direct access inside the bulk leading to weight loss of 95% of PCL/CHT 70/30 within 10 days. On the other hand, the degradation kinetics by using lysozyme was quite straightforward despite the increase of macrovoids and porosity.

The bioactivity of 3D scaffolds used in tissue engineering applications depends on density of the available ligands, scaffolds microstructural environment at which specific cell binding occurs. Ligand density is defined by the composition of the scaffolds and the ligand density on the surface is inversely proportional to the surface macrovoid size or proportional to the effective surface area, surface which is exposed for initial attachment of the cells.

The physico-chemical and structural properties of the developed double porous membrane scaffolds, ultimately strongly affected the cellular morpho-functional behaviour and invasion in the polymeric scaffold bulk which is also supported by our previous study [START_REF] Bacchin | Tunable microstructured membranes in organs-on-chips to monitor transendothelial hydraulic resistance[END_REF]. Our results are in line with other studies that show the viability of stem cells affected by geometry and morphological structure of the scaffolds, which modulate the cell shape and cytoskeletal tension thus regulating cell functions [START_REF] Kang | Adipogenesis of murine embryonic stem cells in a three-dimensional culture system using electrospun polymer scaffolds[END_REF]. In our case, the membrane porosity and ESA were responsible of the different morphology displayed by cells on the offer physical, chemical and mechanical cues for hMSCs attachment and proliferation. Specifically, PCL/CHT 80/20 membrane is characterised by topographical, mechanical and degradation properties that promote long-term cell viability in a culture environment where cells can invade and proliferate inside the macrovoids, which are characterized by a microporous structure that modulate mass transfer of molecules. These properties make the membrane suitable to open new possibility for stem cell based tissue engineering (e.g., liver, vessels).

Ultimately, this membrane scaffold can provide a selective cell co-culture model for different tissues where, according to the cellular dimension, cells can invade and proliferate inside the macrovoids and/or can adhere on the surface ensuring the biochemical cross-talk, which is necessary for recapitulating physiological functions. scaffolds. The increase of surface macrovoids size with consequent decrease of ESA% in the blended PCL/CHT membranes (e.g., PCL/CHT 80/20, PCL/CHT 70/30) reduced the cell adhesion on the surface enhancing the invasion of cells into the bulk that assume a more globular shape. Additionally, the increase of CHT wt% in such membranes causes also an increase of micropore size distribution and surface porosity that are responsible of higher irregular surface. We found on PCL/ CHT 80/20 and PCL/CHT 70/30 high percentage of cells entrapped inside the macrovoids that exhibit a spherical morphology. Differently, lower surface porosity conferred more regular and smoother surfaces, that in addition to higher ESA, enhanced the adhesion of hMSCs on the membrane surfaces as occurred on PCL/CHT 100/0 and PCL/CHT 90/ 10 scaffolds. Indeed, cells adhered on the surface of these membranes achieving a wide spreading. The cell invasion in the double porous membrane scaffolds reduced the cell proliferation that was lower on scaffolds with increase of CHT wt%, which lead to the formation of greater macrovoids and a more porous surface. These results are corroborated by previous findings reported by O'Brien et al. [START_REF] O'brien | The effect of pore size on cell adhesion in collagen-GAG scaffolds[END_REF] that shown a strong correlation between the scaffold specific surface area and cell attachment, and by Murphy et al. [START_REF] Murphy | The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering[END_REF] that highlighted the important role played by the scaffold specific surface area on initial cell adhesion.

In our study, the proliferation rate increased in the time consistently to the scaffold degradation, reaching maximum values after 21 days on all membrane scaffolds especially for hMSCs cultured on PCL/CHT 100/0, 90/10 and 80/20. A further evidence is provided by the OUR data that show a similar trend of cell proliferation. In particular, cellular oxygen consumption is a good indicator of cell viability and functions since the oxygen is one of the most important nutrients for cells and play an important role in regulating MSCs function [START_REF] Muschler | Engineering Principles of Clinical Cell-Based Tissue Engineering[END_REF][START_REF] Nuschke | Mesenchymal stem cells/multipotent stromal cells (MSCs) are glycolytic and thus glucose is a limiting factor of in vitro models of MSC starvation[END_REF]. The OUR of the hMSCs cultured on the blends increased in the time and with the PCL wt% coherently with cell adhesion and proliferation data highlighting an enhanced cellular metabolic activity.

Overall the results demonstrated that the scaffolds were able to create a permissive environment for hMSC adhesion and invasion affecting their morphology, proliferation and metabolism and maintaining their viability and integrity. The macrovoids enabled the cell invasion into the membrane and the microporosity ensured an adequate diffusive mass transfer of nutrients and metabolites, which are essential for the long-term maintenance of cell viability and functions.

Conclusions

In this study, we developed new double porous membrane scaffolds consisting of variable macrovoids interconnected through microporous structure. We found a linear change in scaffolds macrovoids size, effective surface area and surface irregularity by changing the PCL/CHT blend, which has a direct impact on their physico-chemical and degradation properties.

Topographical structure of the membrane scaffolds significantly influenced the morpho-functional behavior of hMSCs. Cell viability and proliferation increased by decreasing macrovoid size and by increasing the effective surface area, which is available for specific integrin-ligand interaction. Cells adhered and proliferated on the surface of PCL/CHT 100/0 and PCL/CHT 90/10 membranes, that are characterized by a high effective surface area and small macrovoids. PCL/CHT 80/20 and PCL/CHT 70/30 enabled the entrapment of cells inside the large macrovoids owing to a limited effective surface area. These results provided evidence that the three-dimensional double porous membrane scaffolds

Fig. 1 .

 1 Fig. 1. SEM images: top surface and cross sections of PCL/CHT double porous membranes made by using the track-etched membrane, and with single porosity made without the track-etched membrane.

Fig. 2 .

 2 Fig. 2. Weight loss (%) of PCL/CHT double porous membranes with different time interval (days) by enzymatic degradation in presence of Lipase (Aspergillus oryzaewith 0.112 U/ml) (A) and Lysozyme (0.013 mg/ml) (B). Data statistically significant according to ANOVA followed by Bonferroni t'test (p < 0.05): A) (*) vs 7 days, ( §) vs 7 and 14 days, on the same substrate; (θ) vs PCL/CHT 100/ 0, ( †) vs PCL/CHT 100/0 and 90/10, ( ‡) vs all, at the same day of culture. B) (*) vs 10 days, ( §) vs 10 and 31 days, on the same substrate; ( †) vs PCL/CHT 100/0 and 90/10, ( ‡) vs all, at the same day of culture.

Fig. 3 .

 3 Fig. 3. SEM micrographs of hMSCs after 7 and 21 days of culture on PCL/CHT double porous membranes at different magnification.

Fig. 4 .

 4 Fig. 4. CLSM images of hMSCs after 7 and 21 days of culture on PCL/CHT double porous membranes. Cells were stained for vimentin (green), CD90 (red) and nuclei (blue). Scale bar 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5 .

 5 Fig. 5. Frequency distributions of cell surface area on the PCL/CHT double porous membranes.

Fig. 6 .Fig. 7 .

 67 Fig. 6. Distribution in the z-axis of vimentin (green), CD90 (red), nuclei (blue) and scaffolds (grey) after 7 (a) and 21 (b) days of hMSC culture on PCL/CHT double porous membranes. CLSM images were collected from the surface to the bulk in the z-scan mode at step size intervals of 5 μm. Scale bar 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1

 1 Morphological, physico-chemical and mechanical properties of the PCL/CHT double porous membranes. Data statistically significant according to ANOVA followed by Bonferroni t'test (p < 0.05): (*) vs PCL/CHT 80/20 and 70/30; (θ) vs PCL/CHT 70/30; ( §) vs all; (◊) vs PCL/CHT 100/0 and 90/10; ( ‡) vs PCL/CHT 100/0; ( †) vs PCL/CHT 80/20.

	PCL/CHT ratio	100/0	90/10	80/20	70/30
	Polymer wt (%)	15	14	14	10
	Macrovoids Diameter (μm)	13 ± 4 (surface)	15 ± 5 (surface)	21 ± 11 (surface)	35 ± 25 ◊ (surface)
		22 ± 3 (bulk)	34 ± 10 ‡ (bulk)	27 ± 13 (bulk)	46 ± 4 § (bulk)
	Mean Flow Pore Diameter (μm)	0.02	0.07	0.05	1.55
	Effective Surface Area (%)	88 ± 1.0 *	84 ± 1.0 *	74 ± 2.0 θ	69 ± 2.0
	Porosity (%)	40 ± 1.3	45 ± 1.2	48 ± 1.9 ‡	56 ± 5.0 §
	Thickness (μm)	45 ± 0.9	50 ± 1.0 ‡	60 ± 0.4 §	55 ± 0.6 ◊
	Tensile Modulus E (N/mm 2 )	143.6 ± 14.1 § (dry)	128.2 ± 4.8 † (dry)	125.3 ± 11.8 (dry)	111.9 ± 4.6 † (dry)
		131.6 ± 2.2 § (wet)	58.5 ± 4.1 * (wet)	29.5 ± 0.8 θ (wet)	10.1 ± 2.7 (wet)
	Ultimate Tensile Strenght Rm (N/mm 2 )	8.3 ± 0.7 § (dry)	3.7 ± 0.4 * (dry)	2.4 ± 0.2 θ (dry)	2.1 ± 0.2 (dry)
		8.1 ± 0.2 § (wet)	2.5 ± 0.3 * (wet)	1.3 ± 0.1 θ (wet)	0.5 ± 0.2 (wet)
	Elongation at break ε (%)	127.6 ± 50 § (dry)	13.1 ± 2.2 (dry)	6.9 ± 0.8 (dry)	2.1 ± 0.4 (dry)
		146.9 ± 68.4 § (wet)	17.6 ± 8.8 (wet)	15.8 ± 5.1 (wet)	9.1 ± 1.2 (wet)
	Hydraulic Permeance (m/Pa s 10 -5 )	0.1 ± 0.001	0.11 ± 0.002	0.14 ± 0.003	0.19 ± 0.004

Acknowledgements

The author would like to acknowledge the funding organisation Erasmus mundus doctorate in membrane engineering-5 th Ed. under European commission (EACEA).

Declaration of Competing Interest

The authors declare no conflict of interests.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110493.