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The reconstruction of bones following tumor excision and radiotherapy remains a challenge. Our previous study, performed using polysaccharide-based microbeads that contain hydroxyapatite, found that these have osteoconductivity and osteoinductive properties. New formulations of composite microbeads containing HA particles doped with strontium (Sr) at 8 or 50% were developed to improve their biological performance and were evaluated in ectopic sites. In the current research, we characterized the materials by phase-contrast microscopy, laser dynamic scattering particle size-measurements and phosphorus content, before their implantation into two different preclinical bone defect models in rats: the femoral condyle and the segmental bone. Eight weeks after the implantation in the femoral condyle, the histology and immunohistochemistry analyses showed that Sr-doped matrices at both 8% and 50% stimulate bone formation and vascularization. A more complex preclinical model of the irradiation procedure was then developed in rats within a critical-size bone segmental defect. In the non-irradiated sites, no significant differences between the non-doped and Sr-doped microbeads were observed in the bone regeneration. Interestingly, the Sr-doped microbeads at the 8% level of substitution outperformed the vascularization process by increasing new vessel formation in the irradiated sites. These results showed that the inclusion of strontium in the matrix-stimulated vascularization in a critical-size model of bone tissue regeneration after irradiation.

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.

International audience; Intravenous administration of fibrinolytic drugs is the standard treatment of acute thrombotic diseases. However, current fibrinolytics exhibit limited clinical efficacy because of their short plasma half-lives and might trigger hemorrhagic transformations. Therefore, it is mandatory to develop innovative nanomedicine-based solutions for more efficient and safer thrombolysis with biocompatible and biodegradable thrombustargeted nanocarrier. Herein, fucoidan-functionalized hydrogel polysaccharide submicroparticles with high biocompatibility are elaborated by the inverse miniemulsion / crosslinking method. They are loaded with the gold standard fibrinolytic-alteplaseto direct site-specific fibrinolysis due to nanomolar interactions between fucoidan and P-selectin overexpressed on activated platelets and endothelial cells in the thrombus area. The thrombus targeting properties of these particles are validated in a microfluidic assay containing recombinant P-selectin and activated platelets under arterial and venous blood shear rates as well as in vivo. The experiments on the murine model of acute thromboembolic ischemic stroke support this product's therapeutic efficacy, revealing a faster recanalization rate in the middle cerebral artery than with free alteplase, which reduces post-ischemic cerebral infarct lesions and blood-brain barrier permeability. Altogether, this proof-of-concept study demonstrates the potential of a biomaterial-based targeted nanomedicine for the precise treatment of acute thrombotic events, such as ischemic stroke.

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.

Within the biomaterials proposed for tissue regeneration, synthetic 3D hydrogels that mimic soft tissues possess great potential for regenerative medicine but their poor vascularization rate is usually incompatible with long-term cell survival. Fabrication of biomaterials that promote and/or accelerate vascularization remains nowadays a challenge. In the present work, hydrogels with tubular geometries ranging from 28 to 680 μm in diameter, that correspond to those of human small artery/veins and arterioles and venules, were prepared. The surface of this tubes was coated with proteins of the extracellular matrix assuring the adhesion of endothelial cells in a monolayer. Interestingly, in the case of small diameter channels, polysaccharide-based hydrogels made of neutral pullulan and dextran that do not allow endothelial cell adhesion, were transformed into active materials guiding endothelial cell behavior solely by modification of the internal microarchitecture, without addition of proteins. Under static conditions, endothelial cell adhesion, migration, proliferation and polarization on the hydrogel was induced, without the addition of any extracellular matrix protein or adhesion peptide; this property was found to be directly dependent on the curvature of the internal channels. In the last years, the impact of the geometry of biomaterials to regulate cell behavior has been highlighted paving the way to use non-flat geometries as cues to develop biomaterials to guide tissue regeneration. Here, we report a functional material based on geometrical cues to assure endothelial cell arrangement in tubular vessel-like structures and providing with new pro-vascularizing properties.

Central nervous system (CNS) lesions are a leading cause of death and disability worldwide. Three-dimensional neural cultures in biomaterials offer more physiologically relevant models for disease studies, toxicity screenings or in vivo transplantations. Herein, we describe the development and use of pullulan/dextran polysaccharide-based scaffolds for 3D neuronal culture. We first assessed scaffolding properties upon variation of the concentration (1%, 1.5%, 3% w/w) of the cross-linking agent, sodium trimetaphosphate (STMP). The lower STMP concentration (1%) allowed us to generate scaffolds with higher porosity (59.9 ± 4.6%), faster degradation rate (5.11 ± 0.14 mg/min) and lower elastic modulus (384 ± 26 Pa) compared with 3% STMP scaffolds (47 ± 2.1%, 1.39 ± 0.03 mg/min, 916 ± 44 Pa, respectively). Using primary cultures of embryonic neurons from PGKCre, Rosa26tdTomato embryos, we observed that in 3D culture, embryonic neurons remained in aggregates within the scaffolds and did not attach, spread or differentiate. To enhance neuronal adhesion and neurite outgrowth, we then functionalized the 1% STMP scaffolds with laminin. We found that treatment of the scaffold with a 100 μg/mL solution of laminin, combined with a subsequent freeze-drying step, created a laminin mesh network that significantly enhanced embryonic neuron adhesion, neurite outgrowth and survival. Such scaffold therefore constitutes a promising neuron-compatible and biodegradable biomaterial.

International audience; Early detection of thrombotic events remains a big medical challenge. Dextran-based submicronic particles bearing Gd(DOTA) groups and functionalized with fucoidan have been produced via a simple and green water-in-oil emulsification/co-crosslinking process. Their capacity to bind to human activated platelets was evidenced in vitro as well as their cytocompatibility with human endothelial cells. The presence of Gd(DOTA) moieties was confirmed by elemental analysis and total reflection X-ray fluorescence (TRXF) spectrometry. Detailed characterization of particles was performed in terms of size distribution, morphology, and relaxation rates. In particular, longitudinal and transversal proton relaxivities were respectively 1.7 and 5.0 times higher than those of DOTAREM. This study highlights their potential as an MRI diagnostic platform for atherothrombosis.

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; The present work details the fabrication of electrospun tubular scaffolds based on the biocompatible and unexploited blend of gelatin and polytrimethylene carbonate (PTMC) as a media (middle layer of blood vessel) equivalent for blood vessel regeneration. An attempt to resemble the media stimulated the selection of gelatin as a matrix (substitution for collagen) with the inclusion of the biodegradable elastomer PTMC (substitution for elastin). -The work highlights the variation of electrospinning parameters and its assiduous selection based on fiber diameter distribution and pore size distribution to obtain smooth microfibers and micropores which is reported for the first time for this blend. Electrospun conduits of gelatin-PTMC blend had fibers sized 6-8 μm and pores sized ~100-150 μm. Young's modulus of 0.40 ± 0.045 MPa was observed, resembling the tunica media of the native artery (~0.5 MPa). An evaluation of the surface properties, topography, and mechanical properties validated its physical requirements for inclusion in a vascular graft. Preliminary biological tests confirmed its minimal in-vitro toxicity and in-vivo biocompatibility. MTT assay (indirect) elucidated cell viability above 70% with scaffold extract, considered to be non-toxic according to the EN ISO-10993-5/12 protocol. The in-vivo subcutaneous implantation in rat showed a marked reduction in macrophages within 15 days revealing its biocompatibility and its possibility for host integration. This comprehensive study presents for the first time the potential of microporous electrospun gelatin and PTMC blend based tubular construct as a potential biomaterial for vascular tissue engineering. The proposed media equivalent included in a bilayer or trilayer polymeric construct can be a promising off-shelf vascular graft.

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.

Background Atherosclerotic plaque phenotypes are classified based on the extent of macrophage infiltration into the lesions, and the presence of certain macrophage subsets might be a sign for plaque vulnerability. The mannose receptor (MR) is over-expressed in activated macrophages. Tilmanocept is a tracer that targets MR and is approved in Europe and the USA for the detection of sentinel lymph nodes. In this study, our aim was to evaluate the potential of 111In-labelled tilmanocept for the detection of MR-positive macrophages in atherosclerotic plaques of apolipoprotein E-knockout (ApoE-KO) mouse model. Methods Tilmanocept was labelled with 111In. The labelling stability and biodistribution of the tracer was first evaluated in control mice (n = 10) 1 h post injection (p.i.). For in vivo imaging studies, 111In-tilmanocept was injected into ApoE-KO (n = 8) and control (n = 8) mice intravenously (i.v.). The mice were scanned 90 min p.i. using a dedicated animal SPECT/CT. For testing the specificity of 111In-tilmanocept uptake in plaques, a group of ApoE-KO mice was co-injected with excess amount of non-labelled tilmanocept. For ex vivo imaging studies, the whole aortas (n = 9 from ApoE-KO and n = 4 from control mice) were harvested free from adventitial tissue for Sudan IV staining and autoradiography. Cryosections were prepared for immunohistochemistry (IHC). Results 111In radiolabelling of tilmanocept provided a yield of greater than 99%. After i.v. injection, 111In-tilmanocept accumulated in vivo in MR-expressing organs (i.e. liver and spleen) and showed only low residual blood signal 1 h p.i. MR-binding specificity in receptor-positive organs was demonstrated by a 1.5- to 3-fold reduced uptake of 111In-tilmanocept after co-injection of a blocking dose of non-labelled tilmanocept. Focal signal was detected in atherosclerotic plaques of ApoE-KO mice, whereas no signal was detected in the aortas of control mice. 111In-tilmanocept uptake was detected in atherosclerotic plaques on autoradiography correlating well with Sudan IV-positive areas and associating with subendothelial accumulations of MR-positive macrophages as demonstrated by IHC. Conclusions After i.v. injection, 111In-tilmanocept accumulated in MR-expressing organs and was associated with only low residual blood signal. In addition, 111In-tilmanocept uptake was detected in atherosclerotic plaques of mice containing MR-expressing macrophages suggesting that tilmanocept represents a promising tracer for the non-invasive detection of macrophages in atherosclerotic plaques.

There is a need for new targets to specifically localize inflammatory foci, usable in a wide range of organs. Here, we hypothesized that the cleaved molecular form of CD31 is a suitable target for molecular imaging of inflammation. We evaluated a bioconjugate of D-P8RI, a synthetic peptide that binds all cells with cleaved CD31, in an experimental rat model of sterile acute inflammation. Male Wistar rats were injected with turpentine oil into the gastrocnemius muscle two days before 99mTc-HYNIC-D-P8RI (or its analogue with L-Proline) SPECT/CT or [18F]FDG PET/MRI. Biodistribution, stability study, histology, imaging and autoradiography of 99mTc-HYNIC-D-P8RI were further performed. Biodistribution studies revealed rapid elimination of 99mTc-HYNIC-D-P8RI through renal excretion with almost no uptake from most organs and excellent in vitro and in vivo stability were observed. SPECT/CT imaging showed a significant higher 99mTc-HYNIC-D-P8RI uptake compared with its analogue with L-Proline (negative control) and no significant difference compared with [18F]FDG (positive control). Moreover, autoradiography and histology revealed a co-localization between 99mTc-HYNIC-D-P8RI uptake and inflammatory cell infiltration. 99mTc-HYNIC-D-P8RI constitutes a new tool for the detection and localization of inflammatory sites. Our work suggests that targeting cleaved CD31 is an attractive strategy for the specific in vivo imaging of inflammatory processes.

A polyol method was used to obtain ultrasmall ZnO nanoparticles (NPs) doped with iron ions and coated with a low molecular weight fucoidan in order to perform in vivo MR and ex vivo fluorescence imaging of athrothrombosis. During the synthesis, the early elimination of water by azeotropic distillation with toluene allowed us to produce NPs which size, determined by XRD and TEM, decreased from 7 nm to 4 nm with the increase of iron/zinc ratios from 0.05 to 0.50 respectively. For the highest iron content (NP-0.50) NPs were evidenced as a mixture of nanocrystals made of wurtzite and cubic phase with a molar ratio of 2.57:1, although it was not possible to distinguish one from the other by TEM. NP-0.50 were superparamagnetic and exhibited a large emission spectrum at 470 nm when excited at 370 nm. After surface functionalization of NP-0.50 with fucoidan (fuco-0.50), the hydrodynamic size in the physiological medium was 162.0 &plusmn, 0.4 nm, with a corresponding negative zeta potential of &minus, 48.7 &plusmn, 0.4 mV, respectively. The coating was evidenced by FT-IR spectra and thermogravimetric analysis. Aqueous suspensions of fuco-0.50 revealed high transverse proton relaxivities (T2) with an r2 value of 173.5 mM&minus, 1 s&minus, 1 (300 K, 7.0 T) and remained stable for more than 3 months in water or in phosphate buffer saline without evolution of the hydrodynamic size and size distribution. No cytotoxic effect was observed on human endothelial cells up to 48 h with these NPs at a dose of 0.1 mg/mL. After injection into a rat model of atherothrombosis, MR imaging allowed the localization of diseased areas and the subsequent fluorescence imaging of thrombus on tissue slices.

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.

Tissue plasminogen activator (tPA) is the sole approved therapeutic molecule for the treatment of acute ischemic stroke. Yet, only a small percentage of patients could benefit from this life-saving treatment because of medical contraindications and severe side effects, including brain hemorrhage, associated with delayed administration. Here, a nano therapeutic agent is realized by directly associating the clinical formulation of tPA to the porous structure of soft discoidal polymeric nanoconstructs (tPA-DPNs). The porous matrix of DPNs protects tPA from rapid degradation, allowing tPA-DPNs to preserve over 70 % of the tPA original activity after 3 h of exposure to serum proteins. Under dynamic conditions, tPA-DPNs dissolve clots more efficiently than free tPA, as demonstrated in a microfluidic chip where clots are formed mimicking in vivo conditions. At 60 min post treatment initiation, the clot area reduces by half (57 + 8 %) with tPA-DPNs, whereas a similar result (56 + 21 %) is obtained only after 90 min for free tPA. In murine mesentery venules, the intravenous administration of 2.5 mg/kg of tPA-DPNs resolves almost 90 % of the blood clots, whereas a similar dose of free tPA successfully recanalize only about 40 % of the treated vessels. At about 1/10 of the clinical dose (1.0 mg/kg), tPA-DPNs still effectively dissolve 70 % of the clots, whereas free tPA works efficiently only on 16 % of the vessels. In vivo, discoidal tPA-DPNs outperform the lytic activity of 200 nm spherical tPA-coated nanoconstructs in terms of both percentage of successful recanalization events and clot area reduction. The conjugation of tPA with preserved lytic activity, the deformability and blood circulating time of DPNs together with the faster blood clot dissolution would make tPA-DPNs a promising nanotool for enhancing both potency and safety of thrombolytic therapies.

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.

International audience; Injection of recombinant tissue plasminogen activator (rt-PA) is the standard drug treatment for thrombolysis. However, rt-PA shows risk of hemorrhages and limited efficiency even at high doses. Polysaccharide-poly(isobutylcyanoacrylate) nanoparticles functionalized with fucoidan and loaded with rt-PA were designed to accumulate on the thrombus. Fucoidan has a nanomolar affinity for the P-selectin expressed by activated platelets in the thrombus. Solid spherical fluorescent nanoparticles with a hydrodynamic diameter of 136 ± 4 nm were synthesized by redox radical emulsion polymerization. The clinical rt-PA formulation was successfully loaded by adsorption on aminated nanoparticles and able to be released in vitro. We validated the in vitro fibrinolytic activity and binding under flow to both recombinant P-selectin and activated platelet aggregates. The thrombolysis efficiency was demonstrated in a mouse model of venous thrombosis by monitoring the platelet density with intravital microscopy. This study supports the hypothesis that fucoidan-nanoparticles improve the rt-PA efficiency. This work establishes the proof-of-concept of fucoidan-based carriers for targeted thrombolysis.

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.

Vascular grafts made of synthetic polymers perform poorly in cardiac and peripheral bypass applications. In these applications, chitosan-based materials can be produced and shaped to provide a novel scaffold for vascular tissue engineering. The goal of this study was to evaluate in vitro the mechanical properties of a novel chitosan formulation to assess its potential for this scaffold. Two chitosan-based hydrogel tubes were produced by modulating chitosan concentration. Based on the standard ISO 7198:1998, the hydrogel tubes were characterized in vitro in terms of suture retention strength, tensile strength, compliance, and burst pressure. By increasing chitosan concentration, suture retention value increased to reach 1.1 N; average burst strength and elastic moduli also increased significantly. The compliance seemed to exhibit a low value for chitosan tubes of high concentration. By modulating chitosan concentration, we produced scaffolds with suitable mechanical properties to be implanted in vivo and withstand physiological blood pressures.

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 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.

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.