3D-Printed Osteoinductive Polymeric Scaffolds with Optimized Architecture to Repair a Sheep Metatarsal Critical-Size Bone Defect.

The reconstruction of critical-size bone defects in long bones remains a challenge for clinicians. A new osteoinductive medical device is developed here for long bone repair by combining a 3D-printed architectured cylindrical scaffold made of clinical-grade polylactic acid (PLA) with a polyelectrolyte film coating delivering the osteogenic bone morphogenetic protein 2 (BMP-2). This film-coated scaffold is used to repair a sheep metatarsal 25-mm long critical-size bone defect. In vitro and in vivo biocompatibility of the film-coated PLA material is proved according to ISO standards. Scaffold geometry is found to influence BMP-2 incorporation. Bone regeneration is followed using X-ray scans, µCT scans, and histology. It is shown that scaffold internal geometry, notably pore shape, influenced bone regeneration, which is homogenous longitudinally. Scaffolds with cubic pores of ≈870 µm and a low BMP-2 dose of ≈120 µg cm-3 induce the best bone regeneration without any adverse effects. The visual score given by clinicians during animal follow-up is found to be an easy way to predict bone regeneration. This work opens perspectives for a clinical application in personalized bone regeneration.

Cytocompatibility / Antibacterial Activity Trade-off for Knittable Wet-Spun Chitosan Monofilaments Functionalized by the In Situ Incorporation of Cu2+ and Zn2.

The wet spinning of cytocompatible, bioresorbable, and knittable chitosan (CTS) monofilaments would be advantageous for a variety of surgical applications. The complexation capacity of chitosan with Cu2+ or Zn2+ can be leveraged to enhance its antibacterial activity, but not at the expense of cytocompatibility. In this work, a wet-spinning process was adapted for the in situ incorporation of Cu2+ or Zn2+ with chitosan dopes to produce monofilaments at different drawing ratios (τtot) with various cation/glucosamine molar ratios, evaluated in the fibers (rCu,f and rZn,f). Cytocompatibility and antibacterial activity of wet-spun monofilaments were, respectively, quantified by in vitro live-dead assays on balb 3T3 and by different evaluations of the proliferation inhibition of Staphylococcus epidermidis (Gram+) and Escherichia coli (Gram-). Knittability was tested by a specific tensile test using a knitting needle and evaluated with an industrial knitting machine. It was found that rCu,f = 0.01 and rZn,f = 0.03 significantly increase the antibacterial activity without compromising cytocompatibility. Wet spinning with τtot = 1.6 allowed the production of knittable CTS-Cu monofilaments, as confirmed by knitting assays under industrial conditions.

Bone regeneration in both small and large preclinical bone defect models using an injectable polymer-based substitute containing hydroxyapatite and reconstituted with saline or autologous blood.

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.

In vitro comparison of three percutaneous atrial septal defect closure devices for endothelialisation and haemocompatibility.

BACKGROUND: Percutaneous device closure of atrial septal defect (ASD) is the gold-standard treatment, but several delayed complications may occur as a result of incomplete device endothelialisation. AIMS: In this in vitro study, we compared three ASD closure devices [Nit-Occlud® ASD-R (device 1); Hyperion™ ASDO (device 2); and Amplatzer™ Septal Occluder (device 3)] in terms of the endothelialisation process, using human endothelial progenitors cells (EPCs), and haemocompatibility. METHODS: EPCs from umbilical cord blood were extracted, cultured and characterised. Device samples were seeded with 100,000 cells/cm2. EPC adhesion was investigated at 3 and 24hours, and EPC proliferation was monitored, which allowed longitudinal follow-up (days 1-12). Haemocompatibility of device samples was assessed using a complement C3a assay and platelet and coagulation activation. RESULTS: With regard to EPC adhesion and proliferation, no statistically significant differences were found between the three devices. We observed for each device a significant time-dependent EPC proliferation, appearing at day 8 for devices 2 and 3 and day 10 for device 1. No complement or platelet activation occurred within 15minutes of contact with devices. However, there was minimal activation of coagulation for the three devices. CONCLUSIONS: In this in vitro study we showed that, despite the three ASD occluders having different device designs and coatings, adhesion and proliferation of human endothelial cells was similar for all devices. This should be further confirmed by similar studies including shear stress forces and anti-thrombotic treatments.

Pullulan/dextran/nHA macroporous composite beads for bone repair in a femoral condyle defect in rats.

The repair of bone defects is of particular interest for orthopedic, oral, maxillofacial, and dental surgery. Bone loss requiring reconstruction is conventionally addressed through bone grafting. Depending on the size and the location of the defect, this method has limits and risks. Biomaterials can offer an alternative and have features supporting bone repair. Here, we propose to evaluate the cellular penetration and bone formation of new macroporous beads based on pullulan/dextran that has been supplemented with nanocrystalline hydroxyapatite in a rat model. Cross-linked beads of 300-500 µm diameters were used in a lateral femoral condyle defect and analyzed by magnetic resonance imaging, micro-computed tomography, and histology in comparison to the empty defects 15, 30, and 70 days after implantation. Inflammation was absent for both conditions. For empty defects, cellularisation and mineralization started from the periphery of the defect. For the defects containing beads, cellular structures filling out the spaces between the scaffolds with increasing interconnectivity and trabecular-like organization were observed over time. The analysis of calcified sections showed increased mineralization over time for both conditions, but was more pronounced for the samples containing beads. Bone Mineral Density and Bone Mineral Content were both significantly higher at day 70 for the beads in comparison to empty defects as well as compared with earlier time points. Analysis of newly formed tissue around the beads showed an increase of osteoid tissue, measured as percentage of the defect surface. This study suggests that the use of beads for the repair of small size defects in bone may be expanded on to meet the clinical need for a ready-to-use fill-up material that can favor bone formation and mineralization, as well as promote vessel ingrowth into the defect site.

A nano-hydroxyapatite--pullulan/dextran polysaccharide composite macroporous material for bone tissue engineering.

Research in bone tissue engineering is focused on the development of alternatives to allogenic and autologous bone grafts that can stimulate bone healing. Here, we present scaffolds composed of the natural hydrophilic polysaccharides pullulan and dextran, supplemented or not with nanocrystalline hydroxyapatite particles (nHA). In vitro studies revealed that these matrices induced the formation of multicellular aggregates and expression of early and late bone specific markers with human bone marrow stromal cells in medium deprived of osteoinductive factors. In absence of any seeded cells, heterotopic implantation in mice and goat, revealed that only the composite macroporous scaffold (Matrix + nHA) (i) retained subcutaneously local growth factors, including Bone Morphogenetic Protein 2 (BMP2) and VEGF165, (ii) induced the deposition of a biological apatite layer, (iii) favored the formation of a dense mineralized tissue subcutaneously in mice, as well osteoid tissue after intramuscular implantation in goat. The composite scaffold was thereafter implanted in orthotopic preclinical models of critical size defects, in small and large animals, in three different bony sites, i.e. the femoral condyle of rat, a transversal mandibular defect and a tibial osteotomy in goat. The Matrix + nHA induced a highly mineralized tissue in the three models whatever the site of implantation, as well as osteoid tissue and bone tissue regeneration in direct contact to the matrix. We therefore propose this composite matrix as a material for stimulating bone cell differentiation of host mesenchymal stem cells and bone formation for orthopedic and maxillofacial surgical applications.

The effect of the co-immobilization of human osteoprogenitors and endothelial cells within alginate microspheres on mineralization in a bone defect.

Bone regeneration seems to be dependant on cell communication between osteogenic and endothelial cells arising from surrounding blood vessels. This study aims to determine whether endothelial cells can regulate the osteogenic potential of osteoprogenitor cells in vitro and in vivo, in a long bone defect, when co-immobilized in alginate microspheres. Alginate is a natural polymer widely used as a biomaterial for cell encapsulation. Human osteoprogenitors (HOP) from bone marrow mesenchymal stem cells were immobilized alone or together with human umbilical vein endothelial cells (HUVEC) inside irradiated, oxidized and RGD-grafted alginate microspheres. Immobilized cells were cultured in dynamic conditions and cell metabolic activity increased during three weeks. The gene expression of alkaline phosphatase and osteocalcin, both specific markers of the osteoblastic phenotype, and mineralization deposits were upregulated in co-immobilized HOPs and HUVECs, comparing to the immobilization of monocultures. VEGF secretion was also increased when HOPs were co-immobilized with HUVECs. Microspheres containing co-cultures were further implanted in a bone defect and bone formation was analysed by muCT and histology at 3 and 6 weeks post-implantation. Mineralization was observed inside and around the implanted microspheres containing the immobilized cells. However, when HOPs were co-immobilized with HUVECs, mineralization significantly increased. These findings demonstrate that co-immobilization of osteogenic and endothelial cells within RGD-grafted alginate microspheres provides a promising strategy for bone tissue engineering.

Subcutaneous-induced membranes have no osteoinductive effect on macroporous HA-TCP in vivo.

Induced Membranes Technique was first described to enhance bone reconstruction of large osseous defects. Previous in vitro studies established their osteoinductive potential, due to the presence of opteoblasts precursors and to high amounts of growth factors contained within. The purpose of this study was to test in vivo the osteoinductive properties of induced membranes on a macroporous HA-TCP in a nonosseous subcutaneous site. Subcutaneous-induced membranes were obtained in 21 rabbits; 1 month later, the membranes were filled with a biphasic calcium phosphate material composed of 75% hydroxyapatite (HA) and 25% beta-tricalcium phosphate associated or not with autograft. Histological and immunohistochemical studies were performed on membrane biopsies. Undecalcified and decalcified sections were qualitatively and quantitatively analyzed. (45)Ca uptake was observed and quantified on the sections using microimager analysis. Dense vascularity was found in the induced membranes. New bone formation was detected in the HA-TCP + autograft samples and increased significantly from 3 to 6 months (p < 0.05). No bone was detected in the biomaterial graft alone in the induced membranes at any time. This study showed that induced membranes placed in a nonosseous site have no osteoinductive properties on a macroporous biphasic calcium phosphate biomaterial.

Mathematical modelling of the distribution of newly formed bone in bone tissue engineering.

New bone formation in bone substitutes is usually investigated by histomorphometric global analysis. This study provides a novel mathematical modelling approach of new bone formation in the use of osteoinductive and functionalized biomaterials for bone tissue engineering. We discuss here the repartition and the probability to get new bone formation inside Biphasic Calcium Phosphate (BCP) loaded with autologous osteogenic cells, functionalized with a cyclo RGD peptide, after implantation in rabbits for 2 and 4 weeks. This local analysis allowed us to complement classical global findings and to demonstrate that after 2 weeks of implantation, the probability of new bone formation was significantly higher in RGD-grafted BCP and that new formed bone was largely distributed from the edge to the centre of the implant. While no significant differences were obtained after 4 weeks of implantation between RGD-grafted and non-grafted materials, distribution of new bone formation inside RGD-grafted materials was significantly more homogeneous as demonstrated by our mathematical modelling approach. In conclusion, local analysis of new bone formation inside macroporous substitutes coupled with mathematical modelling constitutes a potential quantitative approach for the evaluation of the osteoconductive and osteoinductive characteristics of such biomaterials.

Anti-endothelial cell autoantibodies and soluble markers of endothelial cell dysfunction in systemic lupus erythematosus.

OBJECTIVE: To determine if anti-endothelial cell antibodies (AECA) and plasma markers of endothelial cell function are related to disease severity in systemic lupus erythematosus (SLE). METHODS: We measured AECA by human umbilical vein endothelial cell binding, endothelial markers von Willebrand factor, soluble thrombomodulin, and soluble E-selectin by ELISA, and disease severity by SLEDAI and SLICC/ACR in 35 patients with SLE. RESULTS: Despite high levels of IgG AECA (p = 0.001) and von Willebrand factor (p = 0.0007) compared to 21 healthy controls, we found a positive correlation only between IgG AECA and the SLEDAI index (r = 0.393, p = 0.021). CONCLUSION: IgG AECA seem to be related to disease activity in SLE, possibly in a pathogenic role. Conversely, plasma markers of endothelial cell damage seem to be an epiphenomenon and may simply be related to excess inflammation.

Effects of armagnac extracts on human platelet function in vitro and on rat arteriovenous shunt thrombosis in vivo.

INTRODUCTION: The "French paradox", a low cardiovascular mortality compared to the prevalent risk factors, has been attributed to the regular use of red wine, and to the polyphenols it contains. These have among other effects an antioxidant and antithrombotic effect. The French paradox is maximal in southwest France, a region which is the region of production of armagnac, an oak cask aged spirit also rich in polyphenols. METHOD: We tested the effects of a freeze-dried extract of 12-year-old armagnac (EA88) on in vitro human platelet adhesion, and on aggregation induced by collagen or ADP, in the presence or absence of hypoxanthine-xanthine oxidase (HX/XO), at concentrations ranging from 5 x 10(-9) to 5 x 10(-3) g/l, after 15-60 min incubation. We also tested the effects of 2-week oral treatment with 1, 5 and 25 mg/kg EA88 in a rat arteriovenous shunt thrombosis model. RESULTS: EA88 inhibited ADP-induced but not collagen-induced human platelet aggregation in vitro in a concentration- and incubation time-dependent manner, which was greater in the presence of HX/XO. In vivo, giving rats a daily oral dose of EA88 for 2 weeks inhibited thrombus formation in a dose-dependent manner, for doses consistent with the habitual human use of armagnac. CONCLUSION: Armagnac extract EA88 had an antiplatelet and antithrombotic effect that if confirmed in man could contribute to explain the intensity of the French paradox in southwest France.