Raya Bushkalova

Maylis Farno

Christophe Tenailleau

Benjamin Duployer

Daniel Cussac

Angelo Parini

Brigitte Sallerin

Sophie Girod Fullana

Alginate-chitosan PEC scaffolds: A useful tool for soft tissues cell therapy

Alginate

Chitosan Polyelectrolyte complex Cell ther apy Mesenchymal stem cells Blocompatibility Ang!ogenesls

In this study we evaluate macroporous scaffolds made of alginate-chitosan polyelectrolyte complexes (PEC) as tools to optimire the results of soft tissues oell therapy. Cell therapy using mesenchymal stem cells (MSC) bas become attractive for tissue repair and regeneration in a number of acute and chronic injuries. Unfortunately their low retention and/or survival after injection limit their beneficial effects. A biomaterial-assisted im plantation, providi ng oells a thr�mensional (3D) microenvironment is a promising strategy. To this purpose, we designed a family of PEC scaffolds, and studied if they could meet the requirement of such application. Xray tomography showed that ail PEC scaffolds present an interconnected macroporosity, and both rheology and tensile measurements reveal optimized mechanical properties (higher storage moduli and Young moduli) compared to alginate referenoe scaffolds. In vitro assays demonstrated their ability to allow MSC retention (higher than 90%) , lo ng -term viability and RiF2 secretion. Then, we used a skeletal muscle implantation mode! to assess the biological response to scaffolds graft, and showed that they support in vivo vascular formation within the implant-derived tissue. The combination of alginate/chitosan PEC scaffolds architecture and angio genic potential make them appear as interesti ng tools to optimize MSC therapy results in soft tissues.

Introduction

In recent years, cell therapy using Bone Marrow Mesenchymal Stem Cells (BM MSC) has become an attractive tool for tissue repair and re generation in a number of acute and chronic injuries. Ahhough the first studies in the field suggested that the beneficial effects of administered BM MSC might be related to their terminal differentiation in the host oell type, this mechanism of action is controversial. In contrast, it is now well established that BM MSC promote repair of injured tissues through the secretion of a variety of paracrine growth (basic fibroblast growth factor [b FGF), insulin like growth factor 1 [IGFl), hepatocyte growth factor [HGF)) immunomodulatory (interleukine 10 [IL 10)) and an giogenic (vascular endothelial growth factor a [VEGF a)) factors [START_REF] Shologu | Human mesenchymal stem cell secretome from bone marrow or adipose-derived tissue sources for treatment of hypoxiainduced pulmonary epithelial injury[END_REF]. They act as mini pumps secreting locally factors whose properties are particularly attractive for the remission of is chemic injury. In particular, we [START_REF] Mias | Mesenchymal stem cells promote matrix metalloproteinase secretion by cardiac fibroblasts and reduce cardiac ventricular fibrosis after myocardial infarction[END_REF] and others [START_REF] Gnecchi | Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement[END_REF] supported the importance of FGF produced by BM MSC in promoting tissue repair after renal (Mi as et al., 2009) and cardiac [START_REF] Gnecchi | Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement[END_REF] ischemia. Furthermore, we have already shown that MSCs encapsulated in alginate scaffolds maintain their capacity to produoe FGF2 [START_REF] Trouche | Evaluation of alginate[END_REF] and VEGF, FGF2, HGF, EGF, IGF 1 and G CSF after 28 days of encapsulation [START_REF] Ceccaldi | Alginate scaffolds for mesenchymal stem cell cardiac therapy: influence of alginate composition[END_REF][START_REF] Ceccaldi | Elaboration and evaluation of alginate foam scaffolds for soft tissue engineering[END_REF], and retain their initial phenotype and ability to differentiate later under a differentiating medium [START_REF] Trouche | Evaluation of alginate[END_REF][START_REF] Ceccaldi | Alginate scaffolds for mesenchymal stem cell cardiac therapy: influence of alginate composition[END_REF].

The benefits of BM MSC therapy has been demonstrated in the management of ischemic damage of several tissues including heart (Hare et E-mail address: sophie.fullana-girod@univ-t1se3.fr (S. Girod Fullana). 1 These authors provided equal contribution to this work (last author). https://doi.org/10.1016/j.ijpharm.2019.118692

properties of the obtained PEC (in the range of several MPa). The use of PEC formation has more rarely been reported for soft tissue applica tions. In a previous study, we have shown that a combination of process and formulation parameters can lead to a family of alginate chitosan PEC scaffolds suitable for cardiac applications [START_REF] Ceccaldi | Evaluation of polyelectrolyte complex-based scaffolds for mesenchymal stem cell therapy in cardiac ischemia treatment[END_REF]. In this study, we explore if these scaffolds could match the specifications for soft tissue treatment by MSC. We hypothesized that biomaterial design can lead to a construct whose architecture could permit good cell retention and viability combined with angiogenic potential. To this purpose, we designed alginate/chitosan PEC scaffolds with controlled porosity and mechanical properties and demonstrated their ability to allow MSC retention, long term viability and FGF2 secretion in vitro. Next, as a proof of principle, we used a skeletal muscle implantation model to assess the biologic effect of the obtained constructs to support in vivo vascular formation within the implant derived tissue.

Materials and Methods

Materials

Sodium alginate (molecular weight 80,000 120,000; ratio M/ G = 1.56), chitosan (molecular weight 190,000 310,000; 84.6% dea cetylated), HEPES sodium salt, Dulbecco's Phosphate Buffered Saline (DPBS) without calcium chloride and magnesium chloride, as well as antibiotics penicillin streptomycin were purchased form Sigma Aldrich, France. Calcium chloride dihydrate (CaCl₂•2H₂O) and sodium chloride (NaCl) were purchased from VWR. Fetal Medium for cell culture was prepared by supplementing the MEMα with 10% fetal bovine serum and 1% penicillin streptomycin.

Preparation of Alginate/Chitosan Macroporous 3D Scaffolds

Three dimensional alginate/chitosan PEC scaffolds (containing al ginate/chitosan weight ratio of 60/40, 50/50 and 40/60) were pre pared by a freeze drying technique following a method reported pre viously [START_REF] Ceccaldi | Evaluation of polyelectrolyte complex-based scaffolds for mesenchymal stem cell therapy in cardiac ischemia treatment[END_REF]. Briefly, 100 μl of polyelectrolyte solutions or 200 μl of single polymer solution (pure alginate (ratio 100/ 0) or of pure chitosan (ratio 0/100) 1.5% w/w,used as controls) were placed in a 96 well plate, frozen overnight at -20 °C and lyophilized. Constructs were cross linked in an isoosmotic buffer (pH 7.4) con taining 150 Mm NaCl, 0.1 M CaCl₂•2H₂O, and 12.5 mM HEPES during 1 h. Pure chitosan scaffolds were cross linked with NaOH 1 M. All scaffolds were washed 2 times during 1 h in a HEPES buffer (pH 7.4), and lyophilized again. The final scaffolds dimensions, used in all ex periments, were 5 mm diameter × 2.5 mm thickness.

Physico chemical Characterization

Computed X ray Micro tomography (Micro CT)

The micro CT study of samples was carried out on Phoenix Nanotom 180 (GE Sensing, Germany) using the following parameters: 30 kV voltage, 160 microA current, no filter material, 0.25°rotation step, 5 frames as frame averaging, 1440 tomographic projections over a 360°s can angle, 1 s exposure time. A binning 2 × 2 was applied for the slices reconstruction and the resulting voxel size was 11.5 μm 3 . Three di mensional virtual models of scaffolds were obtained using VGStudio MAX 2.1. A region of interest (ROI) was drawn within the reconstructed volume and a threshold was defined to identify the polymeric phase. Then, a morphometric analysis of the ROI was performed to obtain the total porosity and void interconnectivity. Scaffolds' morphologies were analyzed on the basis of 2D X ray tomographic slices using ImageJ (NIH, USA). Calculations were done on a ROI defined on the surface and in the cross section of each scaffold. Feret's diameters were obtained and pore densities were calculated as the total void number/ROI area (n = 10 slices per scaffold). Voids on edges were excluded. intravascular or intraparenchymal injection results into a low rate of retention and/or survival. Intravenous perfusion is hampered by low retention rates within 24 h of delivery, typically < 10%, and a long term survival and engraftment < 1% [START_REF] Mäkelä | Safety and biodistribution study of bone marrowederived mesenchymal stromal cells and mononuclear cells and the impact of the administration route in an intact porcine model[END_REF][START_REF] Malliaras | Cardiac cell therapy: where we've been, where we are, and where we should be headed[END_REF][START_REF] Cheng | Transplantation of bone marrowderived MSCs improves cisplatinum-induced renal injury through paracrine mechanisms[END_REF]. Direct intraparenchymal in jections results in better retention of cells, but leads to poor survival rates, generally < 1% [START_REF] Dec | Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart[END_REF][START_REF] Feygin | Functional and bioenergetic modulations in the infarct border zone following autologous mesenchymal stem cell transplantation[END_REF][START_REF] Morigi | Human bone marrow mesenchymal stem cells accelerate recovery of acute renal injury and prolong survival in mice[END_REF][START_REF] Cheng | Transplantation of bone marrowderived MSCs improves cisplatinum-induced renal injury through paracrine mechanisms[END_REF][START_REF] Wang | Mesenchymal stem cells promote liver regeneration and prolong survival in small-for-size liver grafts: involvement of C-Jun Nterminal kinase, cyclin D1, and NF-κB[END_REF].

To address the need for increased retention and better viability of transplanted cells, a promising strategy is to associate MSCs with a biocompatible material that protects and concentrates them on the damaged area [START_REF] Noronha | Priming approaches to improve the efficacy of mesenchymal stromal cell-based therapies[END_REF]. For tissue engineering applica tions, macroporous scaffolds are of particular interest since they allow improved cell invasion and rapid exchange of nutrients, oxygen and metabolic wastes, while providing the scaffold seeded cells a three di mensional (3D) microenvironment. As a consequence scaffold's design is decisive to optimize cell therapy results. Key requirements include control of scaffold's porosity, mechanical properties, and biocompat ibility. It has been established that pore size and distribution affect the biological performance of the construct by influencing cell morpho genesis [START_REF] Zmora | Tailoring the pore architecture in 3-D alginate scaffolds by controlling the freezing regime during fabrication[END_REF], cell behaviors and host cell colonization [START_REF] Zeltinger | Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition[END_REF][START_REF] Salem | Interactions of 3T3 fibroblasts and endothelial cells with defined pore features[END_REF]. Therefore, the ideal scaffold should present an interconnected porosity with pore sizes compatible with 3D cell culture. In addition, the ideal scaffold should present mechanical properties that not only suit the specific application (ac cording to the mechanical characteristics of the targeted organ) but also allow surgical manipulation during grafting.

Natural polymers seem to be particularly adapted for the elabora tion of cell carrier systems due to both their structural properties and high biocompatibility. Alginates are among the most commonly used biopolymers in regenerative medicine due to their structural simila rities with the extracellular matrix, their gelling properties under con ditions compatible with biological activities (37 °C, pH 7.4 …) and their low cytotoxicity. They are naturally occurring anionic linear (un branched) polysaccharides, which can be extracted from kelp, brown seaweed and some bacteria. In terms of chemical structure, alginates are salts of alginic acid consisting of 1,4 linked β D mannuronic (M) and α L guluronic (G) residues organized in regions of sequential G units (G blocks), sequential M units (M blocks) and G and M units atactically organized. Their sol gel transition properties are based on the formation of a stiff "egg box" structure due to divalent cations se lective binding to the G blocks of two adjacent polymeric chains. A number of studies have demonstrated the benefits of alginate 3D scaf folds seeded with various cell types for the regeneration of heart [START_REF] Zieber | Microfabrication of channel arrays promotes vessel-like network formation in cardiac cell construct and vascularization in vivo[END_REF][START_REF] Dvir | Prevascularization of cardiac patch on the omentum improves its therapeutic outcome[END_REF][START_REF] Leor | Bioengineered cardiac grafts: a new approach to repair the infarcted myocardium?[END_REF], liver [START_REF] Dvir-Ginzberg | Induced differentiation and maturation of newborn liver cells into functional hepatic tissue in macroporous alginate scaffolds[END_REF] and kidney [START_REF] Kim | Renal tissue reconstitution by the implantation of renal segments on biodegradable polymer scaffolds[END_REF]. Although this therapeutic approach seems promising, the use of alginate macroporous scaffolds as implantable devices is challenged by drawbacks such as fragility [START_REF] Ceccaldi | Alginate scaffolds for mesenchymal stem cell cardiac therapy: influence of alginate composition[END_REF] and lack of specific signals for cell attachment [START_REF] Shachar | The effect of immobilized RGD peptide in alginate scaffolds on cardiac tissue engineering[END_REF]. Strategies to improve alginate properties may entail chemical modifications leading to potentially toxic reactive in termediates. One alternative to chemical modification is to associate alginate with a cationic polymer like chitosan, to form polyelectrolyte complexes of opposite charge (PEC) with improved properties. Chitosan is a cationic copolymer of b(1 4) 2 acetamido 2 deoxy b D gucopyr anose and 2 amino 2 deoxy b D glucopyranose, obtained by deacetyla tion of naturally occurring chitin. Chitosan is a biocompatible and biodegradable polymer whose applications in the tissue engineering field include regeneration of bone and articular cartilage [START_REF] Malafaya | Bilayered chitosan-based scaffolds for osteochondral tissue engineering: influence of hydroxyapatite on in vitro cytotoxicity and dynamic bioactivity studies in a specific double-chamber bioreactor[END_REF][START_REF] Ahmed | Mesenchymal stem cell-based tissue engineering strategies for repair of articular cartilage[END_REF], heart [START_REF] Hussain | Functional 3-D cardiac co-culture model using bioactive chitosan nanofiber scaffolds[END_REF][START_REF] Wang | Improved myocardial performance in infarcted rat heart by co-injection of basic fibroblast growth factor with temperature-responsive chitosan hydrogel[END_REF], kidney [START_REF] Gao | The use of chitosan based hydrogel for enhancing the therapeutic benefits of adipose-derived MSCs for acute kidney injury[END_REF], skin [START_REF] Azad | Chitosan membrane as a wound-healing dressing: characterization and clinical application[END_REF], and nervous tissue [START_REF] Gnavi | The use of chitosan-based scaffolds to enhance regeneration in the nervous system[END_REF][START_REF] Zahir | Bioengineering neural stem/progenitor cellcoated tubes for spinal cord injury repair[END_REF].

Use of alginate/chitosan porous scaffolds has been described mainly in the field of bone and cartilage tissue engineering [START_REF] Florczyk | Evaluation of three-dimensional porous chitosan-alginate scaffolds in rat calvarial defects for bone regeneration applications[END_REF][START_REF] Li | Functional recovery after acute intravenous administration of human umbilical cord mesenchymal stem cells in rats with cerebral ischemia-reperfusion injury[END_REF][START_REF] Li | Chitosan-alginate as scaffolding material for cartilage tissue engineering[END_REF][START_REF] Qi | Proliferation and chondrogenic differentiation of CD105-positive enriched rat synovium-derived mesenchymal stem cells in three-dimensional porous scaffolds[END_REF][START_REF] Li | On-site alginate gelation for enhanced cell proliferation and uniform distribution in porous scaffolds[END_REF][START_REF] Shao | Developing an alginate/chitosan hybrid fiber scaffold for annulus fibrosus cells[END_REF]Li et al., 2005) due to the increased mechanical in vitro culture constructs were washed two times with a mix of MEMα and physiological serum (ratio 1/1). Staining solution containing 2 μM ethidium homodimer 3 (necrotic marker measuring nucleus membrane integrity) and 1 μM calcein AM (viability marker measuring the in tracellular esterase activity) was prepared in a mix of MEMα and physiological serum 1/1 to stain dead cells in red and live cells in green. Scaffolds were incubated in the staining solution during 30 min in the dark at room temperature, then washed twice with DPBS and im mediately observed under a confocal microscope Zeiss LSM510 using a 10× objective. The samples were excited with a 488 nm Argon laser and with a 543 nm helium neon laser. The emitted fluorescence was collected using two separate photomultiplier tubes with a BP 500 560 nm filter for calcein detection and a LP 620 nm filter for ethidium homodimer 3 detection. The 3D reconstructions were gener ated using Image J (NIH, USA) from microscopic images where the green and red channel were merged.

Evaluation of Cell Retention After Seeding Within 3D Scaffolds

The efficiency of cell retention after seeding within scaffolds was evaluated by measuring the total lactate dehydrogenase (LDH) activity of cell seeded scaffolds (3D) and of cells left at the bottom of the well (residual) using the Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific). Sixteen hours after seeding, the scaffolds were immersed into a lysis solution and then the total LDH activity was measured ac cording to the manufacturer's instructions. Absorbance was read at 450 nm (specific signal) and 620 nm (background signal) using a plate reading spectrophotometer (Infinite® 200Pro, Tecan Group). After subtraction of the background signal, cell retention efficiency was cal culated as follows:

= - + × Retention efficiency (%) 1 (LDH /(LDH LDH )) 100. residual residual 3D
In addition, a standard curve was established using scaffolds seeded with a known number of MSCs (culture medium and acellular scaffolds served as blank). All measurements were performed in triplicates.

Cell Proliferation in Scaffolds

MSC proliferation in different 3D scaffolds was assessed by mea suring the double stranded genomic DNA content. On 0, 1, 3, 7 and 14 days of culture, the scaffolds were taken out of the culture plate wells, placed in Eppendorf tubes containing one 5 mm stainless steel bead each, and grinded using a mixer mill (Retsch). After that, total DNA was extracted using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma Aldrich) as specified by the manufacturer.

Finally, double stranded DNA content was quantified using the PicoGreen assay (Life Technologies). One hundred microliters of the eluate, containing pure genomic DNA, were transferred to a flat black 96 well plate and the fluorescence was measured at an excitation wa velength of 485 nm and an emission wavelength of 535 nm using a plate reader (Infinite® 200Pro,Tecan Group).

In parallel, two standard curves were established: one curve using purified double strand DNA (DNA diluting buffer was used as blank) and another curve using DNA extracted from scaffolds seeded with a known number of MSCs (acellular scaffolds were used as blank). All measurements were performed in triplicates.

Scanning Electron Microscopy (SEM)

40/60 PEC scaffolds were seeded with MSCs and cultured in vitro in cell culture medium according to the protocol previously described. After 3 days of culture, the samples were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer at 4 °C. The samples were subsequently coated under vacuum with platinum alloy, then immediately flash carbon coated under vacuum. The surfaces of the cell seeded scaffolds were examined in an ESEM Quanta 250 FEG FEI scanning electron micro scope at an accelerating voltage of 5 kV and a magnification of 7000 times.

Swelling

The swelling behaviors of the scaffolds were studied at room tem perature by measuring their weights in a dry (W dry ) and in a wet (W wet ) state using an electronic balance. A dry scaffold was placed in a mesh basket and then immersed in an excess of culture medium. At regular time intervals the basket containing the wet scaffold was taken out of the medium, drained on an absorbent paper and weighed. The swelling ratio was calculated using the following formula:

Swelling ratio (W wet =-W dry )/W dry 2.3.3. Rheological Characterization Rheological measurements were carried out using a stress con trolled rheometer (Rheostress RS75, HAAKE, Germany) with a parallel plate geometry. Test samples matching the diameter of the plate (20 mm) were prepared. After complete rehydration in culture medium, the specimens were carefully laid on absorbent paper to remove excess liquid before lowering the rheometer top plate. Stress sweeps at a constant frequency of 1 Hz were first performed to obtain the linear viscoelastic region for collecting subsequent data. Then, frequency sweeps were performed in the linear viscoelastic regime to determine the storage modulus (G′) and the loss modulus (G″).

Tensile Testing

The scaffolds' tensile properties were evaluated through a uniaxial tensile test performed using a TA XT2 Texture Analyzer (Stable Microsystems, UK) with tensile grips. Specimens were cut as a self de signed dumbbell shape (100 mm total length, 60 mm gage length,8.7 mm gage section), then completely rehydrated in culture medium at room temperature. Excess liquid was removed on tissue paper and the shoulders were fixed between the tensile grips. The specimens were stretched at a constant speed of 2 mm/s and stress strain curves were recorded until rupture. Tensile strength at 30% of elongation were calculated.

In Vitro Assays

Isolation and Culture of Rat Bone Marrow MSCs

Mesenchymal stromal cells were obtained from the femurs of Lewis male rats (Harlan, France) weighing 180 200 g as previously described [START_REF] Ceccaldi | Evaluation of polyelectrolyte complex-based scaffolds for mesenchymal stem cell therapy in cardiac ischemia treatment[END_REF][START_REF] Mias | Mesenchymal stem cells promote matrix metalloproteinase secretion by cardiac fibroblasts and reduce cardiac ventricular fibrosis after myocardial infarction[END_REF]. Briefly, anesthesia was per formed by intraperitoneal injection of pentobarbital (0.1 ml/100 g) before animals were euthanized. Bone marrow was flushed from rat's femur with MEMα supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and centrifuged (300 ×g, 5 min). Then cells were plated in culture flasks (10,000 cells/cm 2 ). After 3 days, non ad herent cells were removed by changing the culture medium, and MSCs were recovered by their capacity to adhere to plastic culture dishes. MSCs are qualified with the expression of surface markers such as CD90/Thy1, CD29//Integrin alpha 9 beta 1, CD106/VCAM 1 and their capacity to differentiate into adipocytes, chondrocytes, and osteocytes in culture. MSCs were then routinely cultured and were used for the experiments between the third and the sixth passage.

MSCs Seeding on Scaffolds

Each scaffold was placed in the well of a 96 well polystyrene plate and seeded by adding drop wise 10 μl of cell suspension containing 100,000 MSCs (for in vitro experiments) or 500,000 MSCs (in vivo study) on the top of the scaffold. The plate was centrifuged 1 min at 400 ×g, and then 290 μl of culture medium was added. The constructs were cultured in a 5% CO 2 incubator at 37 °C. For in vitro culture, the medium was changed every 2 3 days.

Live/Dead Assay and Confocal Microscopy

Live/Dead assays were performed using the Viability/Cytotoxicity Assay kit (FluoProbes®, Interchim, France). After 1, 3, 7 and 15 days of Gene GAPDH (rat): Forward primer sequence: 5′ AGGTCGGTGTGA ACGGATTTG 3′; Reverse primer sequence: 5′ TGTAGACCATGTAG TTGAGGTCA 3′. Gene FGF2 (rat): Forward primer sequence: 5′ GTGTGTGCGAACC GGTACCT 3′; Reverse primer sequence: 5′ TATTGGACTCCAGGCG TTCAA 3′.

Western Blot Analysis of Protein Expression

Protein extracts form MSCs cultured in 2D were obtained by cell lysis in a lysis buffer composed of DPBS 1×, 1% NP40 (IGEPAL®), 12 mM sodium deoxycholate, 0.1% SDS, and supplemented with a protease inhibitor. After centrifugation (10 min, 12,000 ×g, 4 °C) the supernatant and the pellet are separated. The pellet is suspended in a Tris SDS lysis buffer (pH 6.8) composed of 0.08 M Trizma® base (Sigma Aldrich), 5% SDS and 10% glycerol. Protein extracts from MSCs cul tured in 3D scaffolds were obtained after a homogenization in a mixer.

In Vivo Assays

3D scaffolds implantation

Animal experimentations were conducted in accordance with the European Communities Council Directive (2010/63/UE) for experi mental animal care and were approved by local animal care and use committees C2EA

122 (Comités D'éthique En Expérimentation Animale). Rats were housed in an air conditioned room (SPF status) with 12 h light and dark cycles in a controlled environment at a tem perature of 22 ± 2 °C and a relative humidity of 45%. The animals occupied ventilated cages (3 animals / cage) with free access to food and water ad libitum. The bedding material is made of poplar litter and the environment is enriched. Furthermore, the animals were observed daily to assess the clinical state and behavior for the welfare related assessments. Concerning, the experimentations, a randomization has been done to elicit each group.

Animals were under general anesthesia (3% isoflurane) and me chanical ventilation. 3D PEC scaffolds were implanted in adult female Lewis rats (200 220 g). After the skin was prepped and sterilized with ethanol, a small incision (about 1.3 cm) was made on the chest. The Pectoralis major muscle was exposed and a small muscle pouch was formed between two sets of muscle fibers using surgical scissors. Animals were randomly divided into 2 groups. The 'Patch' group (n = 8) received a 40/60 PEC scaffold. Sham operated animals (n = 5) were subjected to the same surgical procedure without scaffold im plantation. Finally, the muscle pouch was closed by a stich. The im planted scaffolds had been hydrated for at least 12 h in MEMα under sterile conditions and their final dimensions were 5 mm diameter x 2.5 mm thickness. The surgical procedure was realized under a Zeiss OPM1 FC microscope.

C reactive Protein Quantification

Measurements of C reactive protein (CRP) concentrations as an acute phase reactant were performed on blood collected by retro or bital bleeding on anesthetized rats from the two different experimental groups described above using heparinized tubes. Blood samples were centrifuged 15 min at 400 ×g and then the plasma was collected. CRP concentrations in plasma were evaluated using the xMAP technology (Luminex 100™ system, Luminexcorp) with the ProcartaPlex Rat CRP kit (eBioscience).

Histology

Histological analyses were performed on muscle specimens fixed in paraformaldehyde (4% solution in DPBS, pH 7.4) and embedded in paraffin.

For basic histomorphological evaluation, paraffin sections (4 μm thick) were stained with hematoxylin and eosin according to standard protocols and slides were scanned using NanoZoomer Digital Pathology.

For immunofluorescence analyses, paraffin sections were dewaxed, rehydrated and incubated in a Tris EDTA antigen retrieval solution (10 mM Trizma® base, 1 mM EDTA, 0.05% Tween20, pH 9) at 121 °C for 3 min. After a permeabilization step (0.5% Triton 100×), nonspecific antigen sites were saturated for 30 min with a DPBS solution containing 2% of goat serum and 1% of bovine serum albumin. Sections were stained overnight with a mouse monoclonal anti α SMA (clone 1A4, Sigma Aldrich, 1:1000, 4 °C) and a polyclonal rabbit anti VWF (A0082, Dako, 1:200, 4 °C). Antibody detection was performed using Alexa Fluor® 568 goat anti mouse and AlexaFluor® 488 goat anti rabbit (Life Technologies, 1:200, 30 min). Cell nuclei were stained by DAPI (Sigma Aldrich, 0.05 μg/ml, 10 min). Finally, sections were mounted with Fluoromount mounting medium (Sigma Aldrich).

Immunostained slides were photographed using a confocal Zeiss LSM 780 microscope. Blood vessel density (number of vessels per mm 2 ) was determined by combining counting of α SMA positive structures and morphological analysis. At least five randomly selected images of the implanted area stained by a SMA fluorescent antibody were quan tified for each animal.

Statistical Analyses

Statistical analyses were performed using GraphPad Prism 4 (Prism GraphPad, San Diego, CA). Results are expressed as mean ± SEM. Statistical comparisons between two unpaired groups were performed using the t test (for data following a Gaussian distribution) or the Mann Whitney test (for data not following a Gaussian distribution). For the plasmatic CRP quantification, before after comparisons were performed using the Wilcoxon matched pairs test. Comparisons between more than two groups were performed using a one way ANOVA with Tukey's post test or using a Kruskall Wallis test with the Dunnett's post test (in case of a non Gaussian distribution). The influence of two factors was determined using two way ANOVA with Bonferroni's post test. A value of p < .05 was considered significant.

2.4.7. RNA Extraction and Real time Quantitative Reverse Transcription PCR (RT qPCR)

Total RNA was isolated from MCSs cultured in 2D or in 3D. Cell pellets or scaffolds were placed in Eppendorf tubes containing buffer RLT and 10% of β mercaptoethanol and then grinded using a mixer mill (Retsch) in the presence of 5 mm stainless steel bead. After that, total RNA extraction was performed using the RNeasy Mini Kit® (Quiagen) as specified by the manufacturer. RNA concentration was determined by measuring the absorbance at 260 nm in a NanoDrop® ND 1000 spec trophotometer.

RNA (300 ng per assay tube) was reverse transcribed to cDNA using the RT SuperScript II reverse transcriptase (Invitrogen). RT qPCR was performed on StepOne+ (Applied Biosystems) using SYBRR® Premix EX Taq™ (Tli RNaseH Plus), Rox Plus (TaKaRa Bio) for detection of rat FGF2 and rat GAPDH (glyceraldehyde 3 phosphate dehydrogenase). The reaction assay mix contained 3 ng of cDNA (1:10 diluted template cDNA), 0.2 μM of primers (Eurogentec) and the SYBR® Premix Ex Taq II, ROX Plus for a final volume of 10 μl per well. Quantification of the housekeeping rat cDNA GAPDH was used as an internal control.

The absence of contaminants was checked by RT qPCR assays of negative control samples in which the Superscript II was omitted. Melting curve analysis was performed to ensure purity of the PCR products. Relative quantification was determined using the compara tive CT method with data normalized to GAPDH. Experiments were performed in duplicate and results were treated with the StepOne Software v2.4 (Applied Biosystems). The sense and antisense primers used are:

Results

Alginate/Chitosan 3D PEC Scaffolds Physico chemical Characterization

3D PEC Scaffolds Porous Architecture

Three different PEC scaffold types with a constant alginate con centration (1.5% w/w) and containing 60/40, 50/50 and 40/60 algi nate/chitosan ratios (w/w) were prepared. In parallel, control scaffolds composed of pure alginate (1.5% w/w, defined as 100/0 alginate/ chitosan ratio) or with pure chitosan (1.5% w/w, defined as 0/100 alginate/chitosan ratio) were prepared according to the same proce dure.

Fig. 1 shows micro CT 2D slices reconstructions of alginate/chitosan PEC scaffolds in comparison with alginate and chitosan ones, and Table 1 summarizes porosity quantification data. All scaffolds were found to present a homogeneous morphology characterized by a highly interconnected macroporous structure. Morphometric analysis showed that all PEC scaffolds presented 100% interconnected pores and a total porosity equal or above 80%, significantly higher than 0/100 S (chit osan scaffolds; total porosity of 63%). The reconstructed scaffolds' slices reveal bigger pores and denser walls for PEC scaffolds in comparison with reference ones. This can be attributed to alginate/chitosan PEC presence, distributed within the scaffolds.

Concerning pore sizes, measurements on micro CT slices showed average pore sizes between 170 and 201 μm for PEC scaffolds. They exhibited larger pore sizes than reference scaffolds, with a significant difference with chitosan reference 0/100 S. Pore density, spanning from 17 to 10 pores per squared millimeter, significantly decreased with raising the chitosan ratio within the PEC formulations (Table 1).

3D PEC Scaffolds Swelling Behavior

With the goal to determine the time needed by the scaffolds to re hydrate after MSC deposit, their swelling kinetics have been studied. The obtained dried scaffolds readily absorbed culture medium and swelled quickly. Their swelling kinetics, presented in Fig. 2, show that within the first 5 min of rehydration the scaffolds have reached their maximal swelling ratio, which remained stable afterwards. All scaffolds preserved their morphology after rehydration and no scaffold de gradation was observed within the 2 h of the experiment. However, PEC scaffolds' swelling ratios were significantly lower and present less variability than those measured for alginate and chitosan reference scaffolds, probably due to the stable ionic interactions between chitosan and alginate chains.

3D PEC Scaffolds Viscoelastic Properties

Swollen scaffolds' rheological properties were studied in dynamic conditions in their linear viscoelastic zone at a shear rate of 10 Pa (Fig. 3). For all scaffolds the storage modulus (G′) was higher than the loss modulus (G″) over a large frequency zone (between 10 and 0.01 Hz), which indicates the existence of viscoelastic gels with pre dominant elastic behavior. In addition, the existence of a plateau for G′ and G″ in this frequency range reveals the formation of well structured hydrogels. G′ values of PEC scaffolds increased proportionally to chit osan ratio; they are typically 10 to 12 times higher than G′ of reference alginate scaffold. For 50/50 S and 40/60 S, the values of storage modulus at 0.5 Hz were even higher than values for reference chitosan scaffold 0/100 S (50/50: 17058 Pa, 40/60: 24767 Pa, 0/100: 13833 Pa).

3D PEC Scaffolds Mechanical (Tensile) Properties

Mechanical behaviors of the PEC and reference scaffold were eval uated in swollen state using a uniaxial tensile test (Fig. 4). We observed that for all scaffolds elongation is more or less linear until break and that the linear region is longer when chitosan content is higher. In addition, the higher the chitosan percentage, the neater the break. PEC scaffolds presented increased Young's moduli compared to control al ginate scaffold, as expected, due to the synergistic interactions between the opposite charged polymers.

MSC Seeded Alginate/Chitosan 3D PEC Scaffolds: In Vitro Assays

MSC Viability, Retention and Proliferation Within Alginate/Chitosan 3D PEC Scaffolds

3D scaffolds were seeded with 100,000 MSCs and examined for cell viability, retention, and proliferation.

Cell viability was evaluated by confocal imaging 1, 3, 7 and 15 days post seeding using the Live/Dead® staining. Volume reconstructions of sequential z plane images, starting from the scaffold surface, are pre sented in Fig. 5. We observed that MSC viability was preserved during the 15 days of in vitro culture, as jugged by the cells' bright green fluorescent staining and the virtually absent red staining. Furthermore, no significant difference was found in cell viability within the different types of scaffolds during the whole in vitro culture period. On the contrary, substantially higher cell densities were observed within PEC scaffolds compared with control alginate scaffolds. Indeed, within PEC scaffolds cell density was quite similar to that observed within chitosan scaffold 0/100 S, which is known to favor cell adhesion. Moreover, within PEC scaffolds cells presented a particular 3D distribution where they formed clusters seeming strongly related to the formation and 3D distribution of polyelectrolyte complexes (previously observed in micro CT).

Seeding efficiency was measured by quantifying the total LDH ac tivity of cell seeded scaffolds and of cells left at the bottom of the well within 16 h of cell deposition. Our results show that the seeding effi ciency was very high, above 90%, for all scaffold types (Fig. 6A). Within PEC scaffolds the seeding procedure reproducibility was greater com pared to control alginate and chitosan scaffolds.

Cell proliferation was measured over 2 weeks by quantifying the amount of genomic DNA per scaffold (Fig. 6B). After one day of 3D culture, MSC number within 40/60 S was maintained identical to the number of cells used for seeding (95%), but not within control scaffolds (minus 50% in 100/0 S and minus 30% in 0/100 S). At the end of the two week period, cell number within 40/60 S was maintained similar to the number of initially seeded cells. In contrast, within 100/ S and 0/ 100 S cell number globally decreases all over the experience. At every time point, cell number within 40/60 S was significantly superior to that in 100/0 S and remained close and even higher to that within 0/ 100 S. The cell loss between day 3 and day 7 within 40/60 S was fol lowed by an increase in cell number by day 14 when it reached its SEM imaging at high magnification revealed the cellular mor phology on the 40/60 S surface three days after seeding (Fig. 7). At this time point the matrix is densely populated with fairly round shaped cells, with a diameter of about 3 μm, presenting numerous fibrillar extensions connecting them to the support (cell matrix interactions) and to neighbouring cells (cell cell interactions). (Fig. 7A). A closer view (Fig. 7B; zoomed area) shows that these cells adhered to the matrix by mean of microvillus extensions of the cytoplasm forming larger "feet" for adhesion to the scaffold. Cells were tightly caught with a fiber network that we believe results from cell surface proteins' in teractions with the 3D scaffold components. Cell cell interactions were also noted via microvillus extensions between neighbouring MSCs.

Taken together, the in vitro experimental results indicate that PEC formation endows new properties of 3D scaffolds which are beneficial for MSC culture on the long term compared with alginate and chitosan alone. Indeed, 40/60 S favors not only cell viability, but also cell ad hesion and proliferation. Therefore, we selected this formulation for further tests.

FGF2 MSC Secrelion Ability Within 40/60 PEC Scaffolds

We investigated the FGF2 secretion in 3D culture conditions by quantification in the culture medium of MSC seeded within 40/60 PEC 3D scaffolds. Analyses were performed 24 h after seeding and results were compared to the amount measured in 20.

We performed Western blot analyses on whole cell lysates (Fig. 8A andB) and also checked the expression levels for FGF2 mRNAs by re verse transcription and quantitative PCR (Fig. 8C). We found that FGF2 protein and FGF2 mRNA expressions were slightly higher within 40/60 PEC scaffold (40/60 S) than in 2D culture conditions, thus clearly showing that 40/60 PEC scaffolds permit to maintain and even have a tendency to promote FGF2 MSC secretion ability.

40/60 PEC Scaffold In Vivo Biocompatibility and Vascularization Aftu Implantation

As a proof of concept, we implanted the 40/60 PEC scaffold in a muscle pouch on the pectoral muscle of healthy rats to investigate its biocompatibility and angiogenic properties. Host tissue's reaction and graft' s fate were monitored at 9 and 28 days after implantation.

Ail surgical incisions healed without evidence of infection. No sig nificant differences in white blood cells level were observed between the control group and the acellular scaffold group (data not shown). Inflammation secondary to scaffold grafting was evaluated through the quantification of plasmatic C reactive protein (CRP) and no significant differences were observed in concentrations between the experimental groups along the study (Table 2). As an example, CRP values were 465 ± 12 µg/ml for the control group and 467 ± 68 µg/ml for the acellular scaffold group at day 9. These results show that the animais did not present systemic infection nor infl ammation.

In order to evaluate host's immune reaction, as well as scaffold's engraftment and vascularization, grafts were harvested post mortem and stained with hematoxylin eosin or immunostained for alpha SMA and VWF.

Hematoxylin eosin stained sections on Fig. 9 reveal tissue response to scaffold implantation. The 40/60 PEC 3D scaffold appear well in tegrated within the mu scular tissue 28 days post implantation (Fig. 8A). Grafts' biodegradation gradually progressed over time and the new tissue integrated well with adjacent muscles. At this time point a large number of small functional blood vessels (diameter 15 20 µm) con taining erythrocytes were present between the degrading biomaterial fragments. The fibrous capsule was reduced to a thin (about 15 µm thick) and vascularized interface between the biomaterial and the muscular tissue (arrows on Fig. 9B andC The main limitation of the scaffolds obtained from biopolymers is their Jack of sufficient mechanical properties for handling and im plantation, and their porosity, which is initially too low to allow ce! lular invasion. Different techniques can be used to strengthen biopo lymers' networks, including chemical modifications and crosslinking [START_REF] Oryana | Chemical crosslinking of biopolymeric scaffolds: current knowledge and future directions of crosslinked engineered bone scaffolds[END_REF]. However, these solutions, if they lead to me chanically stronger structures, are often at the expense of biocompat ibility due to the use of solvents, and potentially toxic reaction intermediates or by products.

The use of two of more biopolymers, associated to form inter penetrating networks, complexes, multilayers or fibers is a promising alternative as it does not imply the use of potentially toxic chemicals [START_REF] Luan | Hydrogel based 3D carriers in the application of stem cell therapy by direct injection[END_REF][START_REF] Del Bakhshayesh | Recent advances on biomedical applications of scaffolds in wound healing and dermal tissue engineering[END_REF][START_REF] Li | Chitosan composite scaffolds for articular cartilage defect repair: a review[END_REF][START_REF] Bombaldi De Souzaa | Mechanically-enhanced polysaccharide-based scaffolds for tissue engineering of soft tissues[END_REF]. Among these possibilities the formation of polyelectrolyte complexes (PEC), involving a negatively charged polymer and a positively charged polymer is one of the most attractive strategies in terms of gain in mechanical properties without loss of biocompatibility. Chitosan is often used as the positively charged polymer because of its biocompatibility [START_REF] Periayah | Chitosan: a promising marine polysaccharide for biomedical research[END_REF] and its easily chemically modifiable structure, mainly to increase its solubility [START_REF] Tan | Injectable, biodegradable hydrogels for tissue engineering applications[END_REF]. Concerning the positively charged polymers, the potential choice is greater [START_REF] Wu | Chitosan-based composite hydrogels for biomedical applications[END_REF]. Among possibilities, alginates are frequently used because of their plant origin (unlike col lagen) and their widely recognized biocompatibility [START_REF] Sarker | Alginate utilization in tissue engineering and cell therapy[END_REF][START_REF] Guarino | Alginate processing routes to fabricate bioinspired platforms for tissue engineering and drug delivery[END_REF][START_REF] Bedian | Bio-based materials with novel characteristics for tissue engineering applicationsa review[END_REF].

Different ways of associating alginate and chitosan have been de scribed in the literature: by mixing at room [START_REF] Florczyk | Influence of processing parameters on for mesenchymal stem cell engraftment on solid organ[END_REF][START_REF] Xu | 3D porous chitosan-alginate scaffold stiffness promotes differential responses in prostate cancer cell lines[END_REF][START_REF] Lv | Influence of chitosan oligosaccharide on the gelling and wound healing properties of injectable hydrogels based on carboxymethyl chitosan/ alginate polyelectrolyte complexes[END_REF] or higher temperature [START_REF] Fletcher | Cell-interactive alginate-chitosan biopolymer systems with tunable mechanics and antibody release rates[END_REF], bilayer [START_REF] Banerjee | Alginate-chitosan composite hydrogel film with macrovoids in the inner layer for biomedical applications[END_REF] or layer by layer [START_REF] Silva | Tuning cell adhesive properties via layerby-layer assembly of chitosan and alginate[END_REF] assemblies, crosslinking [START_REF] Baysal | Chitosan/alginate crosslinked hydrogels: preparation, characterization and application for cell growth purposes[END_REF][START_REF] Naghizadeha | Self-crosslinking effect of chitosan and gelatin on alginate based hydrogels: injectable in situ forming scaffolds[END_REF][START_REF] Zhang | Homologous sodium alginate/chitosan-based scaffolds, but contrasting effect on stem cell shape and osteogenesis[END_REF], 3D printing [START_REF] Liu | Preparation and properties of 3d printed alginate-chitosan polyion complex hydrogels for tissue engineering[END_REF], … They lead to biocompatible buildings whose mechanical properties can be variable [START_REF] Francis | An ice-templated, linearly aligned chitosan-alginate scaffold for neural tissue engineering[END_REF][START_REF] Salehi | Alginate/chitosan hydrogel containing olfactory ectomesenchymal stem cells for sciatic nerve tissue engineering[END_REF][START_REF] Lv | Influence of chitosan oligosaccharide on the gelling and wound healing properties of injectable hydrogels based on carboxymethyl chitosan/ alginate polyelectrolyte complexes[END_REF][START_REF] Xu | 3D porous chitosan-alginate scaffold stiffness promotes differential responses in prostate cancer cell lines[END_REF], even up to correspond to the mechanical properties of hard tissues (Li et 2005;[START_REF] Florczyk | Evaluation of three-dimensional porous chitosan-alginate scaffolds in rat calvarial defects for bone regeneration applications[END_REF]. In this context our strategy was to let these biopolymers chemically unmodified, in order to maintain their excellent initial biocompatibility, and to work on the process to obtain a macroporous 3D architecture with improved rheo logical properties compared with reference polymers. By playing on the mixing conditions and the drying process, we have obtained a family of scaffolds whose mechanical resistance can be adapted to that of soft tissues. Without the use of chemical modifications, porogens or time consuming or expensive processes, we have been able to obtain porous structures that allow deep seeding. After encouraging first results in the cardiac field, we here go further in the characterization of this family of biomaterials and the evaluation their potential as scaffolds aimed to cell therapy of soft tissues.

In the first part of this study we performed a thorough study of the relationships between chemical structure, 3D morphology and me chanical properties of scaffolds obtained by mixing alginate with chit osan in different proportions. We aimed at clarifying the physico che mical parameters which are likely to affect material cell interactions in 3D. We used micro CT to determine the impact of the preparation method on the morphology of the obtained scaffolds. The micro CT study showed that the PEC, formed as a result of alginate and chitosan interactions, give way after freeze drying to a highly porous material (Fig. 1) with interconnected pores whose diameters were in the range reported to allow cell seeding and vessel ingrowth [START_REF] Zeltinger | Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition[END_REF][START_REF] Salem | Interactions of 3T3 fibroblasts and endothelial cells with defined pore features[END_REF]. Comparison of the micro CT slices of the different types of scaffolds suggests that the differences in micro architecture are due to the degree of complexation between the two polymers and 3D distribution of the formed PEC in the bulk material. These differences in chemical composition and in 3D structure im pacted the mechanical properties of the scaffolds. Rheological study in dynamic conditions (Fig. 3) highlighted the linear response to cyclic and progressive stimuli. PEC scaffolds presented a visco elastic beha vior with predominant elastic properties over a large frequency range (0.01 to 5 Hz) which demonstrates the efficacy of the preparation process to form highly structured polymeric network. The storage modulus increased with chitosan proportion and for 50/50 S and 40/ 60 S it achieved values greater than that of semi crystalline chitosan gel. Thus, the dynamic analysis at different frequencies highlighted their resistance to strain and elastic behavior. The mechanical test in tension (Fig. 4) showed their capacity to support high level of de formation (up to 50% for 50/50 S) without breaking and preserving a relatively linear response to stress.

The obtained PEC scaffolds stiffness may vary according to alginate/ chitosan ratio. Soft tissues mechanical properties reported in the lit erature have been determined by means of different mechanical testing methods. Tissues elasticities measured by atomic force microscopy span from 0.5 to 1.5 kPa for nerve and liver tissues [START_REF] Flanagan | Neurite branching on deformable substrates[END_REF][START_REF] Georges | Increased stiffness of the rat liver precedes matrix deposition: implications for fibrosis[END_REF] to 5 9 and 8 17 for smooth and skeletal muscle tissues [START_REF] Engler | Surface probe measurements of the elasticity of sectioned tissue, thin gel sand polyelectrolyte multilayer films: pore structure of 3D porous chitosan-alginate polyelectrolyte complex scaffolds[END_REF][START_REF] Berry | Mesenchymal stem cell injection after myocardial infarction improves myocardial compliance[END_REF][START_REF] Collinsworth | Apparent elastic modulus and hysteresis of skeletal muscle cells throughout differentiation[END_REF][START_REF] Engler | Matrix elasticity directs stem cell lineage specification[END_REF]. Few studies have used tensile tests to determine the elastic modulus of biological tissues. Silver et al. have found that in a tensile test the tendon's elastic modulus was 2 8 kPa [START_REF] Silver | Mechanosensing and Mechanochemical Transduction in Extracellular Matrix: Biological, Chemical, Engineering, and Physiological Aspects[END_REF]; Wagenseil et al. have found that the arterial wall presented an elastic modulus of 0.3 to 1 kPa [START_REF] Wagenseil | Vascular extracellular matrix and arterial mechanics[END_REF]. The proposed PEC 3D scaffolds, whose tensile elastic moduli are ranging from 0.5 to 1.5 kPa, present mechanical properties answering those of soft tissues.

Under other experimental conditions the increase in Young's mod ulus obtained by associating alginate and chitosan reaches values Cell retention capacity was evaluated within 16 h of seeding and then cell number within scaffolds was followed during 14 days. Ali scaffolds presented an excellent seeding efficiency (-9()0/4) in the first hours after seeding (Fig. 6A). Such a high level of cell retention is un derstandable because the cells entrapped in the pores of the scaffold had little chance to escape. However, 24 h after seeding, MSC were efficiently retained only within 40/60 S. This sharp drop in cell num bers for 100/0 S and 0/100 S within the first day after seeding could be due to the fact that alginate and chitosan gels are rather fragile physical gels and may be damaged during centrifugation (used for seeding), which entails cell release out of scaffolds.

Over the following days of in vitro culture (Fig. 6B), cell number remained close to the nurnber of seeded cells within 40/60 S not only thanks to efficient cell retention but also to cell proliferation, particu larly noticeable bet ween day 7 and day 14. In contrast, in the reference scaffolds 100/0 S and 0/100 S, cell number globally decreased.

MSC interactions with 3D PEC scaffolds were examined in this study using SEM microscopy (Fig. 7). Our results revealed that MSC seeded within the 40/60 PEC scaffold established anchoring areas with the substrate through cytoplasmic extensions. Moreover, in a previous publication of our team we have shown by mean of actin vinculin staining and confocal microscopy that the alginate/chitosan ratio 40/ 60 fostered cell adhesion, contrary to reference alginate [START_REF] Ceccaldi | Evaluation of polyelectrolyte complex-based scaffolds for mesenchymal stem cell therapy in cardiac ischemia treatment[END_REF]. Taken together, these observations demonstrate that the studied

Conclusion

In this study we showed that alginate chitosan scaffolds are valuable candidates permitting to promote MSC viability and ease of implanta tion for soft tissue cell treatment. We developed a 3D porous scaffold with suitable porous architecture and mechanical properties using naturally derived biomaterials which, combined with MSC, could be used for cell therapy of ischemic disorders. Our strategy is an attractive way to take advantage of the MSC's paracrine effects and is original for the soft tissue engineering field.

scaffold has the potential to provide an appropriate microenvironment for 3D MSC culture and further ensures its biocompatibility.

In addition, we observed in confocal fluorescence microscopy that within PEC scaffolds MSC formed clusters. This cell distribution could be the consequence of the PEC scaffolds' inhomogeneous porosity, ob served in micro CT, in terms of pore size and wall thickness, compared to reference scaffolds. It could also be related to the 3D distribution of the opposite charge PECs generated by mixing alginate and chitosan which create zones favoring cell attachment. Although chitosan is known for its good cell adhesion properties [START_REF] Ceccaldi | Evaluation of polyelectrolyte complex-based scaffolds for mesenchymal stem cell therapy in cardiac ischemia treatment[END_REF], the lack of cell proliferation within 0/100 S could be due to its small pore diameter evidenced in microtomography. Another factor contributing to cell loss could be the progressive degradation of the alginate and chitosan hydrogels over the two week culture period.

Our in vitro experiments demonstrated the drastic beneficial effect of PEC formation on MSC ingrowth: the 40/60 S scaffold provided better MSC retention and favored their proliferation during a 2 week culture. FGF2 quantification in the medium of cell seeded scaffolds showed that this factor is efficiently secreted without major interaction with the biopolymers constituting the scaffold. Moreover, 40/60 PEC scaffolds have a tendency to promote FGF2 MSC secretion ability.

In the second part of this study, we assessed the biocompatibility of the PEC scaffold by intramuscular implantation on healthy rats. Biocompatibility of engineered scaffolds is a critical issue for the long term efficacy of this therapeutic approach. Although a consistent body of literature exists on alginate's and chitosan's biocompatibility upon implantation [START_REF] Guarino | Degradation properties and metabolic activity of alginate and chitosan polyelectrolytes for drug delivery and tissue engineering applications[END_REF], it was necessary to assess the biocompatibility of the scaffolds prepared in this study. Quantification of plasmatic CRP and hematology analyses demonstrated the lack of chronic inflammation secondary to implantation. During the one month follow up (Fig. 9), the scaffold degraded progressively without immune exacerbation. Neovascularization is another critical point which de termines the success of tissue engineering on the long term. As a con sequence, the numerous newly formed blood vessels we observed 1 month after implantation is one more element demonstrating the beneficial interest of the scaffolds we developed for soft tissues cell therapy.

The selected approach allows taking advantage of MSC paracrine secretions (rather than their ability to migrate and integrate the tissue), and concentrating the cells close to the therapeutic target via a 3D scaffold.

The selected biopolymers, alginate and chitosan, can be provided in clinical grade and their well known biomimicry and biocompatibility, unmodified after engineering, ensures the possibility of a rapid transfer of the scaffolds to a clinical evaluation [START_REF] Dornish | Standards and guidelines for biopolymers in tissue-engineered medical products: ASTM alginate and chitosan standard guides[END_REF]. The de scribed scaffolds are easy to produce, with good reproducibly, at rela tively low cost and using commercially available technologies. In their dry form they have a long conservation time life and convenient for off the shelf use.

The biomaterial fabrication makes use of smooth engineering pro cedures, and the scaffold physical characteristics can be designed to improve cell retention, survival, and interactions with the surrounding tissue as well as to facilitate handle during surgical implantation.

Our team has already shown the potential therapeutic properties of biomaterial assisted MSC implantation in kidney and heart [START_REF] Trouche | Evaluation of alginate[END_REF][START_REF] Ceccaldi | Evaluation of polyelectrolyte complex-based scaffolds for mesenchymal stem cell therapy in cardiac ischemia treatment[END_REF]. The scaffolds described here could suit a variety of soft tissues such as skeletal muscle, lung, liver, and kidney thanks to the possibility to play with chemical composition to obtain scaffolds with tailorable stiffness/elasticity and porosity.

  al., 2009; Williams and Hare, 2011; Karantalis et al., 2014 and Bartunek et al., 2013; Mias et al., 2009; Amsalem et al., 2007; Feygin et al., 2007; Perin et al., 2008), kidney (Mias et al., 2008; Qi and Wu, 2013; Reis et al., 2012; Toge! and Westenfelder, 2012), Jung (Souidi et al., 2013), liver (Pan et al., 2012; Chen et al., 2012; Wang et al., 2014), vasculature (Poitevin et al., 2014) and brain (Li et al., 2005; Chopp et al., 2009). In these studies, BM MSC have been delivered by systemic intravenous infusion or injected into the targeted organ. However, data from preclinical and clinical trials suggest that their •Corresponding author at: Laboratoire de Pharmacie Galénique, CIRlMAT, UMR 5085 UPS-INPT-CNRS, Institut Carnot, llquipe "Phosphates, Pharmacotechnie, Biomatériaux", Faculté des Sciences Pharmaoeutiques, 31062 Toulouse cedex 09, Franoe.

Fig. 1 .

 1 Fig. 1. Micro-CT analysis of the scaffolds: 2D micro-CT images of their cross-section. A: reference alginate (100/0-S) and chitosan (0/100-S) scaffolds; B: PEC scaffolds with various alginate/chitosan ratios 60/40 (60/40-S), 50/50 (50/50-S), and 40/60 (40/60-S). The scale bar is 1 mm for both panels (A and B).

Fig. 2 .

 2 Fig. 2. Stability upon rehydration of the scaffolds: swelling ratios of reference and PEC 3D scaffolds as a function of sample immersion time in cell culture medium.

Fig. 4 .Fig. 5 .Fig. 7 .

 457 Fig. 4. Mechanical behavior of the scaffolds in their fully swollen state using a uniaxial tensile test. A: stress versus strain curves of alginate reference scaffold (100/0-S), chitosan reference scaffold (0/100-S) and a 50/50 PEC scaffold (50/50-S); B: Young's moduli in traction of the 3D scaffolds according to alginate/chitosan ratio. *: p ≤ .05, based on two-way Anova analysis.

  ). These results demonstrate the resolution of the inflammatory process and excellent tissue en graftment of 40/60 S. In vivo vascularization of the implanted constructs was analyzed by staining for VWF and alpha SMA to detect endothelial cells and smooth muscle cells. Examination of immunostained sections revealed the presence of blood vessels throughout the granular tissue resulting from the implant's biodegradation. The number of SMA positive vessels/mm 2 was 166 ± 11.4. This result indicates that alginate chitosan PEC 40/60 scaffolds support vascularization, a very important fact. 4. Discussion One of the major challenges in the field of soft tissues cell therapy is to design strategies allowing the administration of stem/progenitor cells taking the maximum advantage of their therapeutic properties by increasing their survival and paracrine activities and decreasing their potential side effects. To that aim, the interest of a scaffold to improve the administration and optimize ce!! survival is now widely recognized. In this field, polymers of natural origin are widely used because of a variety of advantages, such as their good biocompatibility and biode gradability, and the similarity of their networks with the extracellular matrix. According to the previous reports, collagen, gelatin, alginate, chitosan, hyaluronic acid, agarose, and fibrin are the common biopo lymers applied (Asgharia et al., 2017; Newsom et al., 2019; Tiwari et al., 2019; Li et al., 2019; Kaiser and Coulombe, 2015; Perez Estenaga et al., 2018; Rane and Christman, 2011; Perez et al., 2017; Lev and Seliktar, 2018 ; Liu et al., 2019; Saberianpour et al., 2018).

Fig. 8 .

 8 Fig. 8. FGF2 secretion ability of MSC cultured within 40/60 PEC scaffold or in culture plate 24 h post-seeding. A: quantification by Western blot of FGF-2 expression; B: corresponding image from the Western blot; C: expression levels for FGF2 mRNAs by reverse transcription and quantitative PCR.

Fig. 9 .

 9 Fig. 9. hnplantation of a 40/60 PEC scaffold in a muscle pouch on the pectoral muscle of healthy rats: Host tissue's reaction and graft's fate 28 days after im plantation. Optical micrographs of an implanted 40/60 PEC scaffold harvested 28 days after implantation: A: overview; B and C: zoom to the fibrous capsule formed around the implant. M: muscle; S: scaffold. Black arrows indicate newly-formed blood vessels containing erythrocytes. Scale bars correspond to 1 mm for panel A and to 100 µm for panels B and C.

Table 1

 1 Porosity quantification data on micro-CT reconstructions: mean pore diameter, mean pore density, porosity and voids interconnectivity of PEC and reference scaffolds.

	Alginate/chitosan 3D scaffold	100/0-S	60/40-S	50/50-S	40/60-S	0/100-S
	Mean pore diameter (μm)	149 ± 4	170 ± 4	201 ± 5	194 ± 6	77 ± 1
	Mean pore density (mm 2 )	20.4 ± 0.4	16.5 ± 0.6	13.7 ± 0.1	10.2 ± 0.4	19.3 ± 0.4
	Porosity (%)	83	80	82	82	63
	Voids interconnectivity (%)	100	100	100	100	100

Table 2

 2 Plasmatic C-reactive protein (CRP) quantification at D0, D9 and D28 post implantation for control (sham) group and acellular PEC scaffold (40/60-S) group.

	CRP (μg/ml)	D0	D9	D28
	Sham	456.3 ± 21.74	465.4 ± 12.51	505.2 ± 26.41
	40/60-S	511.4 ± 22.76	467.2 ± 68.07	526.2 ± 49.55

Acknowledgments

This work was supported by INSERM and Région Midi Pyrénées grants. We wish to thank the Cellular Imaging Facility (T.R.I. Genotoul Plateform, Toulouse, France) and the FERMAT federation for X ray tomography facilities.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ ence the work reported in this paper.


M-C Illy

M Autillo

L Guerin

D Guillaumont

E Colineau

P Moisy

Hélène Bolvin

C Berthon

Actinides behavior in solution with polyaminocarboxylates cyclic ligands

Keywords: NMR Spectroscopy, DOTA ligand, MOLCAS calculations, magnetic susceptibility, actinides

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

NMR spectroscopy is one of the most relevant and widespread techniques that provides structural information for solid or liquid samples. It can be applied in many fields from small molecule analysis in organic chemistry to large molecule protein analysis in biology. Recently, with the advent of "nuclearized" spectrometers, it has proven useful in nuclear chemistry as well. The presence of an actinide cation, with 5f electrons, generates a modification of the NMR spectrum i.e. a broadening and/or a shift of the chemical shift, which is characteristic of the cation paramagnetism. The structure of the complex in solution can then be deduced by the 1 H NMR spectrum. The total experimental shift (Δ tot ) depends on the paramagnetic shift (δ para ) whose equation can be solved by magnetic susceptibility measurements (equations a and b).

Quantification of the paramagnetic contribution involves measurement of magnetic susceptibilities, accessible by NMR spectroscopy, and determination of two parameters that depends on the electronic configuration of the metallic cation. Bleaney and Golding have determined and calculated these constants for lanthanide cations. For actinide cations (U, Np, Pu), they are unknown but first molar magnetic susceptibilities have been determined (equation b) for the free ion and the Andipicolinate (DPC) complex.

For a better and more complete understanding of the magnetic behavior of 5f electrons, the present study is focused on the comparison between experiment and theory. Two areas of study are undertaken. First, a magnetic susceptibilities study of actinide (IV and VI) complexes in concentrated hydrochloric and nitric acid solutions (up to 9 M) were performed to complete literature data. Second, a study of the influence of the change in ligand symmetry, specifically from DPC to the DOTA ligand, for actinides (III and IV) are ongoing.


Laure Diaz
email: laure.diaz@cea.fr

Jérôme Serp

Pierre Chamelot

Mathieu Gibilaro

Laurent Massot

RARE METAL EXTRACTION & PROCESSING RARE EARTH ELEMENTS II AND PLATINUM GROUP METALS ELECTROCHEMICAL BEHAVIOR OF NEODYMIUM IN MOLTEN CHLORIDE SALTS

•

  Rare earth (RE) metal are commonly used in advanced technologies Uses: ≈ 40% of RE metal RE metal: • Metallothermy: reduction by Ca, Li, Na metal • Electrolysis in molten salt  impossible in aqueous media Europe: storage of waste containing rare earths Rare earth recycling from permanent magnets NdFeB and NiMH batteries by: • Hydro/pyro process (dissolution, selective extraction, electrolysis) • Pyrometallurgical process (FeCl 2 oxidation or chloration and electrolysis) RARE EARTH ELECTROLYSIS • Fluoride media: Industrial process Nd-Fe alloy : electrolysis of Nd 3+ in LiF-NdF 3 -Nd 2 O 3 molten salt on iron cathode at 1050 °C  Liquid alloy Nd-Fe (80-20%) • Chloride media: Electrodeposition leads to low recovery yields (<40%)  Stability of Nd 2+ in chloride salt  Comproportionation reaction: Inactive pyrochemical lab in glove-boxes (< 10 ppm of H 2 O and O 2 ) • Cell: quartz • Salt are stored at 150°C • Salt fusion in an alumina crucible under Ar • Working temperature: 450 °C (LiCl-KCl) and 650°C (pure LiCl) The electrochemical behavior of Nd 3+ depends on the melt composition (chloroacidity) 𝑵𝒅 𝟐+ + 𝟐𝒆 -→ 𝑵𝒅 𝟎 𝑵𝒅 𝟑+ + 𝒆 -→ 𝑵𝒅 𝟐+ Cyclic voltammograms of NdCl 3 (1 wt%) in LiCl-KCl and LiCl melts at ν=100 mV/s, working electrode: W Square wave voltammetry : • First step: 1 electron exchanged • Second step: not well defined  Effect of comproportionation reaction 2𝑁𝑑 3+ + 𝑁𝑑 0 ↔ 3𝑁𝑑 2+ Square wave voltammograms for the reduction of 1 wt% NdCl 3 at a W electrode in LiCl-KCl at 450 °C and LiCl at 650 °C 𝑊 1/2 = 3,52 𝑅𝑇 𝑛𝐹 Lithium underpotential deposition when neodymium salt concentration is increased (Martinez et al. 2012) • Low Nd 3+ concentration (1 wt. %) at low scan rate (< 10mV/s) • High Nd 3+ concentration (> 3 wt. %) at any scan rate This characteristic was also observed for several lanthanide elements (La, Ce) Electrochemical behavior of neodymium in molten chloride salts Laure DIAZ, February 28, 2017 Reduction of NdCl 3 at 450°C in LiCl-KCl with a high argon flow (10 L/h) by electrolysis under constant current (0.15A/cm 2 <J<0.3A/cm²) Low recovery yield < 45% in LiCl-KCl Significant impact of chemical reaction: dissolution of metal despite polarization E OCP is progressively shifted after several electrolysis to the Nd(III)/Nd(II) redox potential: presence of Nd(II) in solution according to Nernst's law Open circuit potential variation during presence of Nd metal in salt Addition Nd metallic ingot in a BN basket to a fresh LiCl-KCl-NdCl 3 salt: similar changes of E OCP are observed E OCP WE is shifted to Nd(III)/Nd(II) system (spectrophotometric studies: Hayashi et al. 2004) E OCP is stable for several weeks Nd(III) signal doesn't completely disappear: equilibrium between E Nd(III)/Nd(II) and E Nd(II)/Nd(0) Square wave voltammograms at 25 Hz for the reduction of LiCl-KCl-NdCl 3 (1 wt%) after metal addition at a W electrode. Dark-purple salt  NdCl 3 and NdCl 2 in solution Hayashi et al. 2004: NdCl 2 : dark-red; NdCl 3 : violet-blue 𝑁𝑑 2+ ↔ 𝑁𝑑 3+ + 1𝑒 - 𝑁𝑑 2+ + 2𝑒 -↔ 𝑁𝑑 0 Anodic SWV: 1 electron exchanged Cathodic SWV: residual Nd(III) reduction + Nd(II) reduction with an exchange of 2 electrons Number of electrons is well defined in these conditions

Acknowledgments:

Guillaume SERVE (technician)

Sylvain GERAN (intern)