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To move towards clinical applications, tissue engineering (TE) should be validated with human primary cells and offer easy connection to the native vascularisation. Based on a sheet-like bone substitute developed previously, we investigated a mesenchymal stem cells/endothelial cells (MSCs/ECs) coculture to enhance pre-vascularisation. Using MSCs from six independent donors whose differentiation potential was assessed towards two lineages, we focused on donor variability and cell crosstalk regarding bone differentiation. Coculture was performed on calcium phosphate granules in a specific chamber during 1 month. MSCs were seeded first then ECs were added after 2 weeks, with respective monocultures as control groups. Cell viability and organisation (fluorescence, electronic microscopy), differentiation (ALP staining/activity, RT-qPCR) and mechanical cohesion were analysed. Adaptation of the protocol to coculture was validated (high cell viability and proliferation). Activity and differentiation showed strong trends towards synergistic effects between cell types. MSCs reached early mineralisation stage of maturation. The delayed addition of ECs allowed for their attachment on developed MSCs’ matrix. The main impact of donor variability could be here the lack of cell proliferation potential with some donors, leading to low differentiation and mechanical cohesion and therefore absence of sheet-like shape successfully obtained with others. We suggest therefore adapting protocols to cell proliferation potentials from one batch of cells to the other in a patient-specific approach.

In liver tissue engineering, cell culture in spheroids is now well recognized to promote the maintenance of hepatic functions. However, the process leading to spheroids formation is time consuming, costly, and not easy to scale-up for further use in human bioartificial liver (BAL) applications. In this study, we encapsulated HepaRG cells (precursors of hepatocyte-like cells) in 1.5% alginate beads without preforming spheroids. Starting from a given hepatic biomass, we analyzed cell differentiation and metabolic performance for further use in a fluidized-bed BAL. We observed that cells self-rearranged as aggregates within the beads and adequately differentiated over time, in the absence of any differentiating factors classically used. On day 14 postencapsulation, cells displayed a wide range of hepatic features necessary for the treatment of a patient in acute liver failure. These activities include albumin synthesis, ammonia and lactate detoxification, and the efficacy of the enzymes involved in the xenobiotic metabolism (such as CYP1A1/2).Impact statementIt has been recognized that culturing cells in spheroids (SPHs) is advantageous as they better reproduce the three-dimensional physiological microenvironment. This approach can be exploited in bioartificial liver applications, where obtaining a functional hepatic biomass is the major challenge. Our study describes an original method for culturing hepatic cells in alginate beads that makes possible the autonomous formation of SPHs after 3 days of culture. In turn, the cells differentiate adequately and display a wide range of hepatic features. They are also capable of treating a pathological plasma model. Finally, this setup can easily be scaled-up to treat acute liver failure.

The elaboration of biomimetic materials inspired from the specific structure of native bone is one the main goal of tissue engineering approaches. To offer the most appropriate environment for bone reconstruction, we combined electrospinning and electrospraying to elaborate an innovative scaffold composed of alternating layers of polycaprolactone (PCL) and hydroxyapatite (HA). In our approach, the electrospun PCL was shaped into a honeycomb-like structure with an inner diameter of 160 μm, capable of providing bone cells with a 3D environment while ensuring the material biomechanical strength. After 5 days of culture without any differentiation factor, the murine embryonic cell line demonstrated excellent cell viability on contact with the PCL-HA structures as well as active colonization of the scaffold. The cell differentiation, as tested by RT-qPCR, revealed a 6-fold increase in the expression of the RNA of the Bglap involved in bone mineralization as compared to a classical 2D culture. This differentiation of the cells into osteoblasts was confirmed by alkaline phosphatase staining of the scaffold cultivated with the cell lineage. Later on, organotypic cultures of embryonic bone tissues showed the high capacity of the PCL-HA honeycomb structure to guide the migration of differentiated bone cells throughout the cavities and the ridge of the biomaterial, with a colonization surface twice as big as that of the control. Taken together, our results indicate that PCL-HA honeycomb structures are biomimetic supports that promotes in vitro osteocompatibility, osteoconduction, and osteoinduction and could be suitable for being used for bone reconstruction in complex situations such as the repair of maxillofacial defects.

The fast and direct detection of small quantities of biological and chemical species is of key importance for numerous biomedical applications. Extensive research has been conducted on nanoelectronic devices that can perform such detection with high sensitivity using silicon nanowires and nanostructures. However, it was recently demonstrated that Si material suffers a lack of long-term stability in physiological environments at nanometer scale [1,2], and is hence not suited for in situ sensing of biological molecules. The results presented here are two important steps toward the realization of core-shell Si-SiC NWFETs for the detection of biomolecules in liquid media. First, we show that SiC NWs exhibit higher chemical stability than Si NWs under physiological conditions. Second, we present the successful carburation of a thin film of Si resulting in a 3.6 nm thin SiC layer.

Background Our team previously designed and validated a new bioartificial liver (BAL) called Suppliver based on a Prismaflex™device, including fluidized bed bioreactors hosting alginate-encapsulated hepatocytes. To ensure correct fluidization within the bioreactor, the beads need to become heavier with the addition of inert glass microspheres.Methods In this study, we assessed the impact of this additional component on the bead production process, bed fluidization, mass transfer and the mechanical properties of the beads, as well as cell viability and basic metabolic function.Results A concentration of 20 mg (1% v/v) of microspheres for 15–20 million cells per milliliter of alginate solution appears to be the best configuration. The filling ratio for the beads in the bioreactors can reach 60%. Four 250-mL bioreactors represent approximately 15% of the hepatocytes in a liver, which is a reasonable target for extracorporeal liver supply.Conclusions Increasing bead density clearly maintained the performances of the fluidized bed with plasma of different compositions, without any risk of release out of the bioreactor. A 1% (v/v)-concentration of microspheres in alginate solution did not result in any alteration of the mechanical or biological behavior. This concentration can thus be applied to the production of large-scale encapsulated biomass for further use of the Suppliver setup in human scale preclinical studies.

Increasing numbers of requests for transplantable organs and their scarcity has led to a pressing need to find alternative solutions to standard transplantation. An appealing but challenging proposal came from the fields of tissue engineering and regenerative medicine, the purpose of which is to build tissues/organs from scratch in the laboratory and use them as either permanent substitutes for direct implantation into the patient’s body, or as temporary substitutes to bridge patients until organ regeneration or transplantation. Using bioartificial constructs requires administration of immunosuppressant therapies to prevent rejection by the recipient. Microencapsulation has been identified as promising technology for immunoisolating biological materials from immune system attacks by the patient. It is based on entrapping cellular material within a spherical semipermeable polymeric scaffold. This latter defines the boundary between the internal native-like environment and the external “aggressive” one. The scaffold thus acts like a selective filter that makes possible an appropriate supply of nutrients and oxygen to the cellular constructs, while blocking the passage for adverse molecules. Alginate, which is a natural polymer, is the main biomaterial used in this context. Its excellent properties and mild gelation ability provide suitable conditions for supporting viability and preserving the functionalities of the cellular- engineered constructs over long periods. Although much remains to be done before bringing microencapsulated constructs into clinical practice, an increasing number of applications for alginate-based microencapsulation in numerous medical areas confirm the considerable potential for this technology in providing a cure for transplant in patients that excludes immunosuppressive therapies.

Bioreactors composed of microstructured PolyDiMethylSiloxane (PDMS) have been fabricated for perfusion culture of various mammalian cells. During the perfusion culture in the bioreactors for one week to two weeks, cells showed good attachment, spreading and reached their confluence over the microchannels of the bioreactors. In addition, cellular productions measured during the culture demonstrated the cell functionality. Although the cellular activity of the cells in culture are still low even compared to in vivo tissue, the conclusions of this work are encouraging toward future tissue engineering based on in vitro propagation and maturation of cells combined with microfabrication technologies.

We designed a sheet-like bone substitute capable of adapting to different geometries and becoming a standard tissue-engineered process for bone surgery. Preosteoblastic cells were seeded on to a monolayer of calcium phosphate granules and cultured in a flat parallelepipedic cell culture chamber for 1 month. From the various diameters of the granules examined, the 80–200 μm group exhibited the most homogeneous performances regarding both biological (cell morphology, viability, differentiation, and simple metabolic activity) and mechanical (cohesion and stress-strain behavior) properties. This sheet was easy to handle after extraction from the culture chamber and showed versatile geometry and flexibility, making it easy to use for surgeons, especially for small defects of the maxillofacial area.

A free streamline wake model based on singularity distribution is proposed in order to treat the flow past an arbitrary curved obstacle with Helmholtz's wake. The slipping condition gives the vortex distribution on the obstacle and the steady evolution condition is written on the first part of the free streamlines in order to find their locations, the geometry of the second part being fixed by an asymptotic study. The validity of the method is judged by comparing results with those obtained by a formulation, to be used as a standard, which encloses conformal mapping and is an adaptation of Levi-Civita's method. Good agreement leads us to envisage extending the method to multi-element systems. Correlatively, we show a coupling procedure with a boundary layer calculation. Applied to the circular cylinder, it allows to bring out the existence of sub-and supercritical ranges. Although the latter is well predicted for the separation angle and the drag coefficient, the former is only approximately approached, with an overestimate of the critical Reynolds number as an immediate consequence.

Purpose Arteriovenous fistulas (AVFs) are created in patients to enable a permanent vascular access for hemodialysis. The AVF causes changes in the hemodynamic conditions leading to possible complications, stenoses being the most common one. Our objective was to compare the effect of treating the stenosed AVF by balloon-angioplasty, whether followed or not with stenting.Methods We considered an AVF presenting an 60% arterial stenosis and simulated the two endovascular treatments using an implicit approach. We then simulated the fluid-structure interactions (FSI) within (i) the patient-specific stenosed AVF, (ii) the AVF after angioplasty, and (iii) the AVF after angioplasty plus stenting with ANSYS Workbench.Results We show that a self-expandable stent does not modify the curvature of the vessel after angioplasty; it only increases the local Young modulus of the stented wall by an order of magnitude. The results of the FSI simulations indicate that the two treatments induce the same hemodynamic conditions: they both reduce the pressure difference across the stenosis, while maintaining the flow distribution downstream of the stenosis. The venous flow rate that has to be guaranteed for hemodialysis is unaltered. Thanks to its large axial flexibility, the self-expandable stent causes at maximum a three-fold increase in the internal wall stresses at peak systole as compared to angioplasty alone.Conclusions By maintaining the vessel lumen shape over time, the stent is likely to reduce the risk of restenosis that can otherwise occur after balloon-angioplasty because of the viscoelastic recoil of the vessel.

Three-dimensional (3D) culture systems have been introduced to provide cells with a biomimetic environment that is similar to in vivoconditions. Among the polymeric molecules available, sodium-alginate (Na-alg) salt is a material that is currently employed in different areas of drug delivery and tissue engineering, because it offers biocompatibility and optimal chemical properties, and its gelation with calcium chloride provides calcium-alginate (Ca-alg) scaffolds with mechanical stability and relative permeability. In this work, four different preparations of Ca-alg beads with varying Na-alg viscosity and concentration were used for a human hepatoma cell line (Huh-7) encapsulation. The effects of Ca-alg bead preparation on structural cell organization, liver-specific functions, and the expression of specific receptors implicated in hepatotropic virus permissivity were evaluated. Hepatic cells were cultured in 500 μm diameter Ca-alg beads for 7 days under dynamic conditions. For all culture systems, cell viability reached almost 100% at day 7. Cell proliferation was concomitantly followed by hepatocyte organization in aggregates, which adopted two different morphologies (spheroid aggregates or multicellular channel-like structures), depending on Ca-alg bead preparation. These cellular organizations established a real 3D hepatocyte architecture with cell polarity, cell junctions, and abundant bile canaliculi possessing microvillus-lined channels. The functionality of these 3D cultures was confirmed by the production of albumin and the exhibition of CYP1A activity over culture time, which were variable, according to Ca-alg bead condition. The expression of specific receptors of hepatitis C virus by Huh-7 cells suggests encouraging data for the further development of a new viral culture system in Ca-alg beads. In summary, this 3D hepatic cell culture represents a promising physiologically relevant system for further in vitrostudies and demonstrates that an adequate encapsulation condition can be selected for each target application in liver tissue engineering, specifically in viral studies.

Objectives: To compare bicarbonate kinetics and acid base status in HD and HDF for the same patient; and to investigate the effect of patient physiologic parameters on these kinetics.Methods: In order to monitor HCO3-kinetics during dialysis, acid-base parameters, pH, blood gases partial pressures, and HCO3-concentrations were recorded during 3 regular dialysis (HD) and 3 on-line post-dilution HDF sessions performed on 12 patients, using same dialysis fluid with a 38 mmol/l HCO3-concentration. HCO3-mass transfers through the hemodialyzers membranes and into the patients were continuously calculated during the sessions from HCO3-concentrations, together with HCO3-dialysance. The “apparent” HCO3-gain was calculated by integrating over time the instantaneous mass transfer from dialyzer and re-infusion fluid to the patient. A second method consisted in calculating the patient apparent bicarbonate space (ABS) and HCO3-mass (ABS times plasma concentration) at beginning and end of session.Results: No significant differences were observed between acid base parameters at the end of HD and HDF sessions. In contrast to urea clearances, HCO3-dialysances decayed with time during sessions from 110 to 140 ml/min to about 40 ml/min after one hour. The net HCO3-gain was taken as the difference between final and initial HCO3-masses. This net gain was in average 63% of apparent gain in HD and 74% in HDF.Conclusions: Uremic acidosis was well corrected without risk of alkalosis. An unexpected result was the continuous decay of bicarbonate dialysance both in HD and HDF during runs.

Bone tissue engineering is a promising solution for patients with bone defects that require reconstruction. This regenerative therapy consists in culturing osteogenic cells on a biodegradable substrate to obtain a bio-hybrid construct that will stimulate bone healing after implantation. This multidisciplinary technology nevertheless requires further development before it can become routine clinical practice. One challenge is to achieve three-dimensional seeding and osteogenic commitment of mesenchymal stem cells on biomaterials under sterile and reproducible conditions. For this purpose, different dynamic culture systems have been developed. This paper reviews recent advances in the field of bioreactors for bone tissue engineering. The purpose of such systems is to improve nutrient delivery to the cells and generate shear stress that may promote cell differentiation into osteoblastic phenotypes. A brief overview of the value of computational fluid dynamics for understanding the cell environment is also provided. Finally, some proposals are made regarding the use of bioreactors as safe and controllable devices that will help commit cells and biomaterials for the regeneration of bone tissue.

This paper proposes an in vitro hemodiafiltration (HDF) model in which the patient is represented by a 2 L bag of fresh heparinized bovine blood circulated by a 4008H monitor through a 0.6 m2hemodialyzer to investigate kinetics of bicarbonate (HCO3-) during online post-dilution HDF. Five tests were carried out, with three ultrafiltration rates, zero (HD test), 30 and 50 ml/min. Blood gases, pH, HCO3-hematocrit and electrolytes were measured with an ABL77 (Radiometer) blood gas analyzer, and HCO3-was simultaneously measured with a biochemistry analyzer. The variation over time of plasma HCO3-concentrations was also calculated using mass conservation and the model of Legallais et al (JMS 168, 2000, 3–15). Agreement between theoretical and measured concentrations was good during the first 25 minutes of each test, corresponding to the time necessary to dialyze the blood. In hemodialysis (HD), there was an HCO3-mass transfer into blood through the membrane due to diffusion which vanished after 40 minutes, while in HDF tests, blood lost HCO3-due to ultrafiltration after 10 minutes. With reinjection, the net HCO3-mass flow rate to the “patient” decayed, from 1.8 mmol/min at t=0 to zero at the end of the test (t=60 min), and was higher in HD than in HDF. “Patient” dialysance, taking into account reinjection, was positive in all tests, and decayed from about 110 ml/min to 40 ml/min at the end of dialysis. These data confirmed that online HDF automatically corrects acidosis without creating alkalosis when HCO3-dialysate concentration is around 30 mmol/L.

Objectives Alginate beads allow cultivation of cells in a 3-dimensional environment. The aim of our study was to assess the influence of a 3-dimensional culture in alginate microbeads, on hepatic cell metabolism.Methods We used 2 types of alginate: low viscosity (LV) and medium viscosity (MV). The hepatic cell line C3A was encapsulated in alginate beads. Cells were cultured for 2 weeks. Using scanning electron microscopy, the morphology of 3D structures and the surfaces of cells were analyzed. Fluidized bed bioartificial liver experiments were performed 24 hours, 7, and 14 days after bead formation.Results Two different cell growth types in alginate beads were observed: channel-like structures and spherical aggregates characteristic of LV and MV alginate, respectively. A significant increase in albumin synthesis was observed in long-term culture. Formation of characteristic hepatic cell microvilli on cell surfaces was observed under scanning electron microscopy for both types of alginate. Prolonged static cultivation of C3A cells within the alginate beads in both types of alginates caused significant increases in albumin production in the fluidized bioreactor.Conclusions Cultivation of the hepatic C3A cells within the alginate microbeads significantly improved bioreactor effectiveness in albumin production. The presence of extensions of cell membranes on the surface of hepatoma cells in 3-dimensional culture within the alginate beads indicated formation of microvilli-like structures characteristic of normal hepatocytes.

This review aims at presenting the dialysis monitors currently present on the French market. After some basics on physiopathology and dialysis therapy, this paper describes the equipments as well as the new technologies recently introduced. These data are collected in a number of technical tables.

Fluidized bed bioartificial liver has been proposed as a temporary support to bridge patients suffering from acute liver failure to transplantation. In such a bioreactor, alginate beads hosting hepatocytes are in continuous motion during at least six hours. After having shown in vitro the functionality of such a device, the present study aims at analyzing the potential mechanical alterations of the beads in the bioreactor, perfused by different surrounding media. Compression experiments are performed and coupled for analysis with Hertz theory. They provide qualitative and quantitative data. The average value of the shear modulus, calculated for the different cases studied varied from 2.4 to 10.4 kPa, and could therefore be considered as a quantitative measure of the beads mechanical properties. From the compression experiments and the estimated values of the shear modulus, we could now evaluate the effect of different operating conditions (fluidization, presence of cells, surrounding medium) on the mechanical behavior of alginate beads. On the one hand, the motion during six hours in the bioreactor does not alter the beads significantly. On the other hand, the presence of different substances in the fluid phase might change their mechanical strength. These results can be considered as new encouragements to use such a device as a bioartificial organ.

Fluidized bed bioreactor with alginate beads may be an alternative to hollow fiber cartridge to host hepatocytes for bioartificial liver purposes. After the bioreactor design and the characterization of fluid mechanics, the present study was aimed at analyzing bi-directional mass transfers of calibrated species between external fluid and empty beads. Static (batch) and dynamic (fluidized bed bioreactor) experimental conditions were analyzed. A simple modelling approach permitted the definition of mass transfer coefficients.The motion of beads within the bioreactor clearly enhanced mass transfer kinetics, but did not alter the amount exchanged. The shear enhanced diffusion coefficient for VitB12 was 20 times higher in the fluidized bed bioreactor than under batch conditions, proving the efficiency of such a device.

Extravascular bioartificial pancreas based on hollow fiber seems to be a promising treatment of diabetes mellitus. However, solutes mass-transport limitations in such a device could explain its lack of success. To determine critical device parameters, we have developed a novel tridimensional model based on finite element method for glucose, insulin, and oxygen diffusion around an islet of Langerhans encapsulated in a hollow-fiber section. A glucose ramp stimulation was applied outside the fiber and diffused to the islet. Concomitantly, a stationary oxygen partial pressure was applied outside the fiber, and determined local oxygen partial pressure on the islet environment. An insulin secretion model stimulated by a glucose concentration ramp and corrected by the local oxygen partial pressure was also implemented. Insulin secretion by the islet was thus computed as a response to glucose signal. The model predictions notably showed that the fiber radius had to be small enough to favor a fast response for insulin secretion and to ensure a maximal oxygen partial pressure in the islet environment. Besides the effect of fiber radius, a better islet oxygenation could be achieved by adjustments on the islet density, i.e., on the fiber length dedicated to a single islet. These hints should allow the future proposal of an optimal design for an implantable bioartificial pancreas. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 576–582, 2002.