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Microbeads consisting of pullulan and dextran supplemented with hydroxyapatite have recently been developed for bone tissue engineering applications. Here, we evaluate the bone formation in two different preclinical models after injection of microbeads reconstituted with either saline buffer or autologous blood. Addition of saline solution or autologous blood to dried microbeads packaged into syringes allowed an easy injection. In the first rat bone defect model performed in the femoral condyle, microcomputed tomography performed after 30 and 60 days revealed an important mineralization process occurring around and within the core of the microbeads in both conditions. Bone volume/total volume measurements revealed no significant differences between the saline solution and the autologous blood groups. Histologically, osteoid tissue was evidenced around and in contact of the microbeads in both conditions. Using the sinus lift model performed in sheep, cone beam computed tomography revealed an important mineralization inside the sinus cavity for both groups after 3 months of implantation. Representative Masson trichrome staining images showed that bone formation occurs at the periphery and inside the microbeads in both conditions. Quantitative evaluation of the new bone formation displayed no significant differences between groups. In conclusion, reconstitution of microbeads with autologous blood did not enhance the regenerative capacity of these microbeads compared to the saline buffer group. This study is of particular interest for clinical applications in oral and maxillofacial surgery.

Freeze-drying is a process of choice to texture hydrogel scaffolds with pores formed by an ice-templating mechanism. Using state-of-the-art microscopies (cryo-EBSD, μCT, CLSM), this work evidences and quantifies the effect of crosslinking and ice nucleation temperature on the porous structure of thin hydrogel scaffolds freeze-dried at a low cooling rate. We focused on a polysaccharide-based hydrogel and developed specific protocols to monitor or trigger ice nucleation for this study. At a fixed number of intermolecular crosslinks per primary molecule (p = 5), the mean pore size in the dry state decreases linearly from 240 to 170 μm, when ice nucleation temperature decreases from -6 °C to -18 °C. When ice nucleation temperature is fixed at -10 °C, the mean pore size decreases from 250 to 150 μm, as the crosslinking degree increases from p = 3 to p = 7. Scaffold infiltration ability was quantified with synthetic microspheres. The seeding efficiency was assessed with MC3T3-E1 individual cells and HepaRG™ spheroids. These data collapse into a single master curve that exhibits a sharp transition from 100 % to 0 %-efficiency as the entity diameter approaches the mean pore size in the dry state. Altogether, we can thus precisely tune the porosity of these 3D materials of interest for 3D cell culture and cGMP production for tissue engineering.

Vascularization of tissue-engineered constructs is critical for proper cell and graft survival. In order to achieve this, pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), are often incorporated into scaffolds by methods that either involve multiple steps or risk compromising protein bioactivity. In this study, we demonstrate a simple approach to incorporate VEGF into polysaccharide electrospun fibers by taking advantage of the interactions between VEGF and sulfated polysaccharide, fucoidan. Pullulan/dextran (P/D) electrospun fibers (diameter ∼500 nm) incorporating fucoidan were fabricated by a one-step electrospinning process. Thereafter, VEGF was loaded onto the scaffolds. By varying the content of the chemical crosslinker, trisodium trimetaphosphate (STMP), from 10 to 12 and 16 wt% (denoted as STMP10, 12 and 16 respectively), the extent of fucoidan incorporation was significantly enhanced (

Organoids production is a key tool for in vitro studies of physiopathological conditions, drug-induced toxicity assays, and for a potential use in regenerative medicine. Hence, it prompted studies on hepatic organoids and liver regeneration. Numerous attempts to produce hepatic constructs had often limited success due to a lack of viability or functionality. Moreover, most products could not be translated for clinical studies. The aim of this study was to develop functional and viable hepatic constructs using a 3D porous scaffold with an adjustable structure, devoid of any animal component, that could also be used as an in vivo implantable system. We used a combination of pharmaceutical grade pullulan and dextran with different porogen formulations to form crosslinked scaffolds with macroporosity ranging from 30 &micro, m to several hundreds of microns. Polysaccharide scaffolds were easy to prepare and to handle, and allowed confocal observations thanks to their transparency. A simple seeding method allowed a rapid impregnation of the scaffolds with HepG2 cells and a homogeneous cell distribution within the scaffolds. Cells were viable over seven days and form spheroids of various geometries and sizes. Cells in 3D express hepatic markers albumin, HNF4&alpha, and CYP3A4, start to polarize and were sensitive to acetaminophen in a concentration-dependant manner. Therefore, this study depicts a proof of concept for organoid production in 3D scaffolds that could be prepared under GMP conditions for reliable drug-induced toxicity studies and for liver tissue engineering.

International audience; Objective: Polysaccharide-based composite matrices consisting of natural polysaccharides, pullulan and dextran supplemented with hydroxyapatite (Matrix-HA) have recently been developed. The principal objective of this study was to evaluate the capacities of this composite material to promote new bone formation in a sinus lift model in the sheep. Secondary objectives were to evaluate in vitro properties of the material regarding cell adhesion and proliferation.Methods: In this report, once such composite matrix was prepared as injectable beads after dispersion in a physiological buffer, and evaluated using a large animal model (sheep) for a sinus lift procedure.Results: In vitro studies revealed that these microbeads (250-550μm in diameter) allow vascular cell adhesion and proliferation of Endothelial Cells (EC) after 1 and 7 days of culture. In vivo studies were performed in 12 adult sheep, and newly formed tissue was analyzed by Cone Beam Computed Tomography (CBCT scanning electron microscopy (SEM) and by histology 3 and 6 months post-implantation. CBCT analyses at the implantation time revealed the radiolucent properties of these matrices. Quantitative analysis showed an increase of a dense mineralized tissue in the Matrix-HA group up to 3 months of implantation. The mineralized volume over total volume after 6 months reached comparable values to those obtained for Bio-Oss® used as positive control. Histological examination confirmed that the Matrix-HA did not induce any long term inflammatory events, and promoted direct contact between the osteoid tissue and lamellar bone structures and beads. After 6 months, we observed a dense network of osteocytes surrounding both biomaterials as well as a newly vascularized formed tissue in close contact to the biomaterials.Significance: In conclusion, the absence of animal components in Matrix-HA, the osteoconductive property of Matrix-HA in sheep, resulting in a dense bone and vascularized tissue, and the initial radiolucent property to follow graft integration offer great promises of this composite material for clinical use.

The present work deals with the fabrication of electrospun tubular scaffold based on in-situ crosslinked blend of gelatin - oxidized carboxymethyl cellulose (OCMC) for vascular tissue engineering. The flow behavior and spinability of the hydrogel despite the in-situ crosslinked gelatin chains evaluated by Raman spectroscopic studies and rheological studies was utilized for electrospinning. The study highlights the tunable pore size and fiber diameter of the nanofibers with the manipulation of electrospinning parameters. With a future perspective of vascular tissue engineering, the electrospinning parameters yielding smooth bead free fibers and maximum magnitude in pore size and fiber diameter as well their homogenous distribution were selected for the fabrication of tubular constructs which is rarely reported. The surface and mechanical properties were evaluated to validate its properties to the native vessel. Biocompatibility was studied in vitro with BALB/c 3T3 cells and in vivo after subcutaneous implantation in rats. MTT assay confirmed its no-toxicity and no abnormal foreign body reaction were observed by 7 and 15days after implantation. Crosslinking with biocompatible crosslinker OCMC has rendered insolubility to gelatin yet making it spinable for electrospinning to fabricate porous, nanofibrous vascular biomaterial.

International audience; (1) Background: Reperfusion injury refers to the cell and tissue damage induced, when blood flow is restored after an ischemic period. While reperfusion reestablishes oxygen supply, it generates a high concentration of radicals, resulting in tissue dysfunction and damage. Here, we aimed to challenge and achieve the potential of a delivery system based on astaxanthin, a natural antioxidant, in attenuating the muscle damage in an animal model of femoral hind-limb ischemia and reperfusion. (2) Methods: The antioxidant capacity and non-toxicity of astaxanthin was validated before and after loading into a polysaccharide scaffold. The capacity of astaxanthin to compensate stress damages was also studied after ischemia induced by femoral artery clamping and followed by varied periods of reperfusion. (3) Results: Histological evaluation showed a positive labeling for CD68 and CD163 macrophage markers, indicating a remodeling process. In addition, higher levels of Nrf2 and NQO1 expression in the sham group compared to the antioxidant group could reflect a reduction of the oxidative damage after 15 days of reperfusion. Furthermore, non-significant differences were observed in non-heme iron deposition in both groups, reflecting a cell population susceptible to free radical damage. (4) Conclusions: Our results suggest that the in situ release of an antioxidant molecule could be effective in improving the antioxidant defenses of ischemia/reperfusion (I/R)-damaged muscles.

Nanogels were prepared in aqueous media without the use of any organic solvent via a simple polyelectrolyte complexation method between aminated pullulan and fucoidan followed by covalent crosslinking with genipin. Homogeneously distributed genipin crosslinked nanogels (G-PECs) were obtained with a mean hydrodynamic diameter of ~155 nm and zeta potential of 0.86 ± 4.35 mV. Their capacity to bind to human activated platelets was evaluated in vitro, as well as their cytocompatibility within human endothelial cells after 1 day of incubation up to 1000 µg/mL of G-PECs (94.56 ± 7.82% of viable cells). Additional hemolysis tests support the biocompatible character of the developed nanosystems (hemolysis rate of 2.09 ± 0.06% for 1000 µg/mL of G-PECs). Under acid conditions, the surface charge of G-PECs was tuned to around ~10 mV allowing miRNA incorporation via electrostatic interactions. G-PECs were able to promote miRNA delivery inside cells, as demonstrated by fluorescence microscopy images of labelled miRNA. With further studies to demonstrate the biological activity of delivered miRNA, these nanogels could be an interesting platform for miRNA-based therapeutics in atherothrombotic-related diseases thanks to the possibility to target over-expressed P-selectin.

The development of new vascular devices requires to study the effects of materials on blood cells and on coagulation, both in vitro and in vivo. In this study, we have developed a new material by grafting dermatan sulfate (DS) from shark skin onto polyethylene terephthalate (PET). We have evaluated the haemocompatibility of PET-DS material in vitro by measuring thrombin generation, plasma recalcification time, hemolytic activity, and platelet adhesion and in vivo with a model of vascular patch in rat abdominal aorta. In vitro, our results have shown that PET-DS is a nonhemolytic material, able to inhibit thrombin generation and platelet adhesion. In vivo studies by Doppler echographic evaluation 20 days after implantation have shown that the PET-DS patch was integrated in the vessel wall and covered by a layer of cells. In conclusion, PET-DS has good haemocompatibility properties and could be a promising tool for vascular surgery. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2001-2009, 2017.

International audience; Nanostructured lipid carriers (NLC) might represent an interesting approach for the identification and targeting of rupture-prone atherosclerotic plaques. In this study, we evaluated the biodistribution, targeting ability and safety of 64Cu-fonctionalized NLC in atherosclerotic mice. 64Cu-chelating-NLC (51.8±3.1 nm diameter) with low dispersity index (0.066±0.016) were produced by high pressure homogenization at tens-of-grams scale. 24 h after injection of 64Cu-chelated particles in ApoE-/- mice, focal regions of the aorta showed accumulation of particles on autoradiography that colocalized with Oil Red O lipid mapping. Signal intensity was significantly greater in aortas isolated from ApoE-/- mice compared to wild type (WT) control (8.95 [7.58, 10.16]×108 vs 4.59 [3.11, 5.03]×108 QL/mm2, P < 0.05). Moreover, NLC seemed safe in relevant biocompatibility studies. NLC could constitute an interesting platform with high clinical translation potential for targeted delivery and imaging purposes in atherosclerosis.

Thrombotic diseases rarely cause symptoms until advanced stage and sudden death. Thus, early detection of thrombus by a widely spread imaging modality can improve the prognosis and reduce mortality. Here, polymer microbubbles (MBs) made of degradable poly(IsoButylCyanoAcrylate) and functionalized with fucoidan (Fucoidan-MBs) were designed as a new targeted ultrasound contrast agent to image venous thrombus. The physicochemical characterizations demonstrate that the MBs with fucoidan surface exhibit a size of 2–6 μm and stability in suspension at 4 °C up to 2 months. MBs exhibit high echogenicity and could be completely burst under high destructive pulse. Flow chamber experiments on activated human platelets show a higher affinity of Fucoidan-MBs than control anionic MBs (CM-Dextran-MBs) under shear stress conditions. In vivo analysis by ultrasound and histological results demonstrate that Fucoidan-MBs are localized in rat venous thrombotic wall, whereas few CM-Dextran-MBs are present. In addition, the binding of Fucoidan-MBs in healthy vein is not observed. Collectively, Fucoidan-MBs appear as a promising functionalized carrier for ultrasound molecular imaging in thrombotic diseases.

Hydrogels are very promising for tissue engineering as they provide scaffolds and a suitable microenvironment to control cell behavior and tissue regeneration. We used a patented method to obtain beads of pullulan/dextran cross-linked with sodium trimetaphosphate (STMP), that were already described for in vivo bone repair. The aim of this study was to provide a comparative analysis of microbeads made of polysaccharides prepared using three different STMP feeding ratio of 1.5, 2.25 or 3 % w/w. The morphology, swelling and biodegradability of these structures were assessed. Mesenchymal stem cells were also seeded to evaluate the cell organization onto the beads. We found that the amount of phosphorus resulting from the cross-linking was proportional to the introduced STMP concentration. An increase of cross-linking decreased the in vitro enzymatic degradability, and also decreased the swelling in PBS or water. The microstructures observed by SEM and confocal microscopy indicated that homogeneous spherical microbeads were obtained, except for the lower cross-linking ratio where the shapes were altered. Beads hydrated in PBS exhibited a mean diameter ranging from 400 to 550 µm with the decrease of STMP ratio. Cells adhered to the surface of microbeads even in the absence of protein coating. Cell viability studies revealed an increase in cell numbers over two weeks for the highest cross-linked beads, whereas the two lowest STMP concentrations induced a decrease of cell viability. Overall, this study demonstrated that pullulan/dextran hydrogels can be designed as microbeads with adjustable physicochemical and biological properties to fulfill requirements for tissue engineering approaches.

International audience; Because cell interactions play a fundamental role for cell differentiation, we investigated the expression of Pannexin 1 and Pannexin 3 in human bone marrow mesenchymal stromal cells (HBMSCs) in a three-dimensional (3D) microenvironment provided by a polysaccharide-based macroporous scaffold. The pannexin (Panx) family consists of three members, Panx1, Panx2, and Panx3. The roles of Panx large-pore ion and metabolite channels are recognized in many physiological and pathophysiological scenarios, but the role of these proteins in human physiological processes is still under investigation. Our study demonstrates that HBMSCs cultured within 3D scaffolds have induced Panx1 and Panx3 expression, compared with two-dimensional culture and that the Panx3 gene expression profile correlates with those of bone markers on mesenchymal stromal cells culture into the 3D scaffold. We showed that Panx1 is involved in the HBMSCs 3D cell-cell organization, as acting on the size of cellular aggregates, demonstrated by the use of Probenecid and the mimetic peptide 10panx1 as specific inhibitors. Inhibition of Panx3 using siRNA strategy shows to reduce the expression of osteocalcin as osteoblast-specific marker by HBMSCs cultured in 3D conditions, suggesting a role of this Panx in osteogenesis. Moreover, we evaluated Panx1 and Panx3 expression within the cellularized scaffolds upon subcutaneous implantation in NOG (NOD/Shi-scid/IL-2Rγnull ) mice, where we could observe a more intense expression in the constructs than in the surrounding tissues in vivo. This study provides new insights on the expression of pannexins in HBMSCs on a 3D microenvironment during the osteogenic differentiation, in vitro and in vivo.

The aim of this study was to functionalize 3D porous cross-linked scaffolds with natural non-animal sulfated polysaccharide fucoidans in order to allow a delivery of vascular endothelial growth factor (VEGF) and potentiate its angiogenic activity. Microporous (20 μm) and macroporous (200 μm) scaffolds were functionalized with low, medium, or high molecular weight fucoidans (named LMWF, MMWF, and HMWF, respectively). In vitro, addition of fucoidans promoted endothelial progenitor cells proliferation in both micro- and macroporous scaffolds. While control scaffolds without fucoidans loaded with VEGF165 (100 ng) showed a fast burst release in PBS during the first 24 h, MMWF significantly reduced the VEGF165 release (p

The potential of electrospun pullulan/dextran (P/D) nanofibers (average diameter = 323 nm) for vascular tissue engineering applications is explored. The mechanical properties of the nanofibers are of the same order of magnitude as that of human arteries (Young's modulus ≈0.88 MPa; tensile strength ≈0.35 MPa). It is demonstrated that the nanofiber topography enables cell adhesion and that the endothelial phenotype is maintained on the nanofibers. Moreover, P/D nanofibers support a stable confluent monolayer of endothelial cells over 14 d. SMCs seeded on nanofibers display similar levels of alpha smooth muscle actin and a lower proliferation rate than cells on 2D cultures. The observations suggest that nanofibers promote a shift to a quiescent contractile phenotype in SMCs.

International audience; Poly(vinyl alcohol) hydrogel (PVA) is a widely used material for biomedical devices, yet there is a need to enhance its biological functionality for in vitro and in vivo vascular application. Significance of surface topography in modulating cellular behaviour is increasingly evident. However, hydrogel patterning remains challenging. Using a casting method, planar PVA were patterned with micro-sized features. To achieve higher patterning resolution, nanoimprint lithography with high pressure and temperature was used. In vitro experiment showed enhanced human endothelial cell (EC) density and adhesion on patterned PVA. Additional chemical modification via nitrogen gas plasma on patterned PVA further improved EC density and adhesion. Only EC monolayer grown on plasma modified PVA with 2 mm gratings and 1.8 mm concave lens exhibited expression of vascular endothelial cadherin, indicating EC functionality. Patterning of the luminal surface of tubular hydrogels is not widely explored. The study presents the first method for simultaneous tubular molding and luminal surface patterning of hydrogel. PVA graft with 2 mm gratings showed patency and endothelialization, while unpatterned grafts were occluded after 20 days in rat aorta. The reproducible, high yield and high-fidelity methods enable planar and tubular patterning of PVA and other hydrogels to be used for biomedical applications.

Predicting acute clinical events caused by atherosclerotic plaque rupture remains a clinical challenge. Anatomic mapping of the vascular tree provided by standard imaging technologies is not always sufficient for a robust diagnosis. Yet biological mechanisms leading to unstable plaques have been identified and corresponding biomarkers have been described. Nanosystems charged with contrast agents and targeted towards these specific biomarkers have been developed for several types of imaging modalities. The first systems that have reached the clinic are ultrasmall superparamagnetic iron oxides for Magnetic Resonance Imaging. Their potential relies on their passive accumulation by predominant physiological mechanisms in rupture-prone plaques. Active targeting strategies are under development to improve their specificity and set up other types of nanoplatforms. Preclinical results show a huge potential of nanomedicine for cardiovascular diagnosis, as long as the safety of these nanosystems in the body is studied in depth.

International audience; New tools for molecular imaging and targeted therapy for cardiovascular diseases are still required. Herein, biodegradable microcapsules (MCs) made of polycyanoacrylate and polysaccharide and functionalized with fucoidan (Fuco-MCs) are designed as new carriers to target arterial thrombi overex-pressing P-selectin. Physicochemical characterizations demonstrated that microcapsules have a core-shell structure and that fucoidan is present onto the surface of Fuco-MCs. Furthermore, their sizes range from 2 to 6 µm and they are stable on storage over 30 d at 4 °C. Flow cytometry experiments evidenced the binding of Fuco-MCs for human activated platelets as compared to MCs (mean fluorescence intensity: 12 008 vs. 9, p < 0.001) and its absence for non-activated platelets (432). An in vitro flow adhesion assay showed high specific binding efficiency of Fuco-MCs to P-selectin and to activated platelet aggregates under arterial shear stress conditions. Moreover, both types of micro-capsules reveal excellent compatibility with 3T3 cells in cytotoxicity assay. One hour after intravenous injection of microcapsules, histological analysis revealed that Fuco-MCs are localized in the rat abdominal aortic aneurysm thrombotic wall and that the binding in the healthy aorta is low. In conclusion, these micro-capsules appear as promising carriers for targeting of tissues characterized by P-selectin overexpression and for their molecular imaging or treatment.

Objectifs L’identification retrospective d’un syndrome coronarien aigu resolu pose un probleme diagnostique. Certaines molecules d’adhesion, telles que les selectines, persistent a la surface de l’endothelium apres la fin de l’ischemie. Elles representent de ce fait une empreinte de cet episode dans ce qui est communement appele la memoire ischemique. Le but de cette etude est d’evaluer l’utilite du fucoidane radiomarque au 99mTechnetium, un agent ciblant la P- et la E-selectine, dans la detection retrospective d’episodes d’ischemie-reperfusion sur un modele de rat. Materiels et methodes Des episodes transitoires d’ischemie myocardique ont ete generes par ligature coronaire chez des rats mâles Wistar. La duree de l’ischemie variait entre 5 et 20 min et le delai apres reperfusion entre 2 et 24 heures. Les acquisitions TEMP/TDM ont ete realisees 2 heures apres injection de 70 MBq de 99mTc-fucoidane. L’analyse scintigraphique a consiste en la determination visuelle de l’existence d’une fixation focale myocardique et en une analyse semi-quantitative par calcul du rapport de fixation du cœur sur le compartiment sanguin (RCS). Les animaux ont par la suite ete sacrifies et leur cœur explante, congele et sectionne pour analyse autoradiographique (quantification par calcul du rapport entre la region myocardique a risque et une region myocardique saine) et histologique/immunohistologique. Resultats Pour un delai constant de 2 heures apres reperfusion, une fixation myocardique de 99mTc-fucoidane a ete observee pour des durees d’ischemie de 20 minutes (8/8 rats, RCS : 2,4 ± 0,9 [2,0-4,7]), 10 minutes (10/10 rats, RCS : 3,6 ± 1,7 [1,5-7,5]) et 5 minutes (8/12 rats, RCS : 2,6 ± 1,8 [1,9-7,1]). Sur l’autoradiographie, le rapport de fixation entre les zones a risque et saines etait de 4,5 ± 1,9 [3,2-9,3] a 20 min, 7,4 ± 3,1 [2,8-12,1] a 10 min et 5,6 ± 2,1 [3,4-8,6] a 5 min. Les regions fixant intensement le 99mTc-fucoidane coincidaient topographiquement avec l’expression endotheliale de P-selectine. Apres 6 heures de reperfusion, la fixation de 99mTc-fucoidane etait detectee apres 20 minutes d’ischemie (7/7, RCS : 3,0 ± 0,6 [2,8-4,5]), 10 min d’ischemie (8/8 rats, RCS : 2,9). Conclusions Cette etude demontre que des episodes d’occlusion coronaire brefs suivis de reperfusion de durees variables, allant jusqu’a 24 heures, sont associes a l’expression endotheliale de P- et E-selectine, detectables in vivo par la TEMP/TDM au 99mTc-fucoidane. Ces donnees soutiennent la possibilite d’une utilisation clinique du 99mTc-fucoidane dans la detection de la memoire ischemique.

The potential of electrospun pullulan/dextran (P/D) nanofibers (average diameter = 323 nm) for vascular tissue engineering applications is explored. The mechanical properties of the nanofibers are of the same order of magnitude as that of human arteries (Young's modulus ≈0.88 MPa; tensile strength ≈0.35 MPa). It is demonstrated that the nanofiber topography enables cell adhesion and that the endothelial phenotype is maintained on the nanofibers. Moreover, P/D nanofibers support a stable confluent monolayer of endothelial cells over 14 d. SMCs seeded on nanofibers display similar levels of alpha smooth muscle actin and a lower proliferation rate than cells on 2D cultures. The observations suggest that nanofibers promote a shift to a quiescent contractile phenotype in SMCs.