Hybrid Artificial Organs Research Articles

Bioprinting on 3D Printed Titanium Scaffolds for Periodontal Ligament Regeneration.

The three-dimensional (3D) cell-printing technique has been identified as a new biofabrication platform because of its ability to locate living cells in pre-defined spatial locations with scaffolds and various growth factors. Osseointegrated dental implants have been regarded as very reliable and have long-term reliability. However, host defense mechanisms against infections and micro-movements have been known to be impaired around a dental implant because of the lack of a periodontal ligament. In this study, we fabricated a hybrid artificial organ with a periodontal ligament on the surface of titanium using 3D printing technology. CEMP-1, a known cementogenic factor, was enhanced in vitro. In animal experiments, when the hybrid artificial organ was transplanted to the calvarial defect model, it was observed that the amount of connective tissue increased. 3D-printed hybrid artificial organs can be used with dental implants, establishing physiological tooth functions, including the ability to react to mechanical stimuli and the ability to resist infections.

Hepatic differentiation of mouse embryonic stem cells in a three-dimensional culture system using polyurethane foam

Embryonic stem (ES) cells are a type of pluripotent stem cell line isolated from the inner cell mass of blastocysts and characterized by an almost unlimited self-renewal capacity and differentiation potential in vitro into multiple cell lineages. Therefore the use of ES cells has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional matured cells from ES cells in large quantities. In this study, we applied polyurethane foam (PUF)/spheroid culture, which enables spontaneous spheroid formation and mass cultivation of cultured cells, to mouse ES cells for hepatic differentiation. Mouse ES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of the PUF within 1 d. To induce hepatic differentiation, specific growth factors were added to the culture medium. Mouse ES cells proliferated by day 20, and high cell density (about 1.0 x 10(8) cells/cm(3)-PUF) was achieved. Differentiating ES cells expressed endodermal-specific genes, such as alpha-fetoprotein, albumin and tryptophan 2,3-dioxygenase. The activity of ammonia removal of mouse ES cells per unit volume of the module was detected by day 21 and increased with culture time. Maximum expression levels were comparable to those of primary mouse hepatocytes. Mouse ES cells could express liver-specific functions at high level because of the high cell density culture and hepatic differentiation. These results suggest that the PUF/spheroid culture method could be useful to develop mass differentiation cultures.

Hepatic Differentiation of Embryonic Stem Cells in HF/Organoid Culture

Objective The use of embryonic stem cells (ES cells) has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional mature cells from ES cells in large quantities. We applied HF/organoid culture, where cultured cells formed cylindrical multicellular aggregates (organoids) in the lumen of hollow fibers, to mouse and cynomolgus monkey ES cells for hepatic differentiation. Materials and Methods ES cells were injected into hollow fibers. The hollow fibers were centrifuged to induce organoid formation and cultured in medium including factors for hepatic differentiation. To determine the characteristics of cells in the bundle, we evaluated gene expression and liver-specific functions. Results ES cells immobilized inside hollow fibers proliferated and formed cylindrical organoids. In mouse ES cell cultures, the expression of mRNAs of hepatocyte-specific genes increased with culture time. Ammonia removal activity detected at 15 days increased with culture time. Albumin secretion activity detected at 12 days increased by 21 days. In cynomolgus monkey ES cell cultures, ES cells showed spontaneous ammonia removal functions. The maximum levels of these functions per unit volume of the hollow fibers were roughly comparable to those of primary hepatocyte-organoids. Conclusions ES cells differentiated into hepatocyte-like cells using the organoid culture technique. The results indicated that the combination of ES cells and an organoid culture technique was useful to obtain mature hepatocytes.

Study on basic technology and evaluation method for hybrid artificial organs

Study on basic technology and evaluation method for hybrid artificial organs

Lentiviral vector: a useful tool for transduction of human liver endothelial cells.

Endothelial cells play multiple roles in pathophysiologic processes and are increasingly being recognized as target cells of gene therapy. Lentiviral vectors derived from human immunodeficiency virus type 1 have an ability to infect both dividing and nondividing cells and currently receive a great deal of attention as an innovative tool for transduction of target cells. The purpose of the present work was to evaluate the efficacy of a lentiviral vector for transducing human liver endothelial cells (HLECs) in vitro. For the present study, a pseudotyped lentiviral vector encoding a green fluorescent protein (GFP) gene, LtV-GFP, was generated by means of FuGENE 6 method and allowed to infect HLECs. Approximately 95% of HLECs were positive for GFP expression after LtV-GFP infection at a multiplicity of infection of 10. Notably, LtV-GFP transduced HLECs had stable and long term GFP expression, showed gene expression of endothelial markers including CD 34, factor VIII, flt-1, KDR/flk-1 and HGF, and maintained in vitro angiogenic potential in a Matrigel assay to the same extent as primarily cultured HLECs. These findings provide evidence that lentivirus based gene delivery is an efficient tool for transduction of endothelial cells that could be considered for cell and gene therapies and hybrid artificial organs.

Polymeric biomaterials synthesized by radiation techniques – current studies at IARC, Poland

AbstractFor over 15 years the Institute of Applied Radiation Chemistry in Lodz, Poland, has been involved in studies on new polymeric biomaterials. Some of the technologies developed employ radiation techniques for the synthesis of crosslinked products (mainly hydrogels), for surface modification and for sterilization. An example of a mature technology of this kind is hydrogel wound dressings, now being produced on a large scale and available on the market in many Central European countries. Another advanced project is a hydrogel system for healing endometrium cancer by precise, local drug delivery. Preliminary clinical tests with over 70 patients have been completed with very good results.Current research projects, besides further development of the above mentioned systems, include: a hydrogel‐based system for local, controlled drug delivery; polymer materials for encapsulation of living cells and for the construction of hybrid artificial organs; a new approach to the synthesis of polymeric material for intervertebral disc implant; temperature‐sensitive membranes and supports for cell cultures; hydrogel phantoms for low‐dose, three‐dimensional radiation dosimetry; degradation‐resistant nanogels and microgels for biomedical purposes (e.g. synovial fluid substitute); hydrogel‐based dietary products and adjustment of the molecular weight of biopolymers by ultrasound and ionizing radiation. Copyright © 2003 John Wiley & Sons, Ltd.

Historical perspectives of hybrid hepatic assist devices: tissue sourcing, immunoisolation, and clinical trial.

A hybrid hepatic assist device using canine liver tissues was developed and clinically applied 38 years ago. However, for many years practical hybrid hepatic assist devices were not clinically introduced owing to the many difficulties encountered in employing cultured hepatocytes. These problems include: (1) maintenance of viable cultured cells, (2) maintenance of normal hepatocyte function with these cells, (3) elimination of toxic substances generated by non-viable cultured and/or stored cells, (4) elimination of immunological factors generated by cultured cells and by the patient, and (5) difficulties of the biocompatible immunological barrier for cells against the patient. Fortunately, recent progress in apheresis and biomaterial technologies enable us to isolate cultured cells immunologically and yet maintain effective metabolic functions for the patient. These technologies generate an immunological barrier of a hybrid hepatic assist device for the patients. Proper adsorption columns developed for apheresis procedures enable us to remove the toxic substances released by non-viable cells. Recent development of oxygen-carrying macromolecules enable us to provide sufficient oxygen supply to the cultured cells and to maintain their normal cellular function, not only during the cultured period of time, but also during their actual clinical application. Together with the advancement of cell culture technologies, including the proper cultured environments and cellular seeding environments, these technologies, primarily developed for therapeutic apheresis, should be able to provide more effective and safe hybrid artificial organs.

In vivo tissue engineering: dreams for the future

Both tissue-engineered organs and hybrid artificial organs are considered to be candidates for the satisfaction of future hopes. The author has been engaged in developing vascular prostheses-neointima formed on vascular prostheses was a product of tissue engineering in vivo. On the basis of the author's experiences, the advantages of in vivo tissue engineering technologies for future artificial organs and dreams for the future are described. The use of primitive young cells is an important point; they proliferate easily and can differentiate along with their environment. They also synthesize various kinds of cytokines and growth factors, which are useful for tissue engineering. Combined use of undifferentiated cells and growth factors can induce organogenesis, so we can aim for the creation of new organs in vivo. The rejuvenation phenomenon of cells, i.e., blastogenesis, is also useful. Extracellular matrix can control cell differentiation, migration, and formation of cell communities, so we must learn to modify extracellular matrices. We humans have an extremely important legacy in our bodies, although we do not know how to use it. We have accumulated many genes during our evolution, and such genes do not become extinct. Therefore we have many genes from our primitive ancestors. These primitive creatures had an extremely strong capability for tissue repair, and we must find a way to make practical application of these dormant traits. In the future human beings will be able to learn how to control and apply them for in vivo tissue engineering technologies.

A new trend in hybrid artificial organs

A new trend in hybrid artificial organs

Designing biomaterials to direct biological responses.

We have set forth a design strategy for creating biomimetic materials that direct the formation of tissue surrounding implants or regeneration within porous scaffolds. Our studies have established that heterogeneous mimetic peptide surfaces (MPS) containing both the -RGD- (cell-binding) and-FHRRIKA- (putative heparin-binding) peptides, unique to BSP, in the ratio of 75:25 (MPS II) or 50:50 (MPS III) proved to be more biologically relevant and specific for RCO cell function. The initial response of human osteoblast-like cells to these surfaces was mediated by the collagen (alpha 2 beta 1) and vitronectin receptors (alpha v beta 3), whereas the vitronectin receptor alone dominated longer-term events (> 30 min). MPS II and III surfaces enhanced cell spreading and long-term events such as mineralization of the extracellular matrix compared to homogenous peptide surfaces and controls. Furthermore, extensive mineralization of the ECM deposited by RCOs occurred when the peptide was coupled to an interfacial interpenetrating polymer network (IPN) that resisted protein deposition (i.e., non-specific adsorption) and fouling. Work on thermo-reversible P(NIPAAm-co-AAc) hydrogels demonstrated the ability to create materials that can be delivered to the body in a minimally invasive manner and support tissue regeneration. These hydrogels can be modified to incorporate biofunctional components such as the biomimetic peptides, theoretically enhancing their ability to foster tissue regeneration. These results suggest that biomaterials can be engineered to mimic ECM components of bone (e.g., various organs) by grafting peptides in the appropriate ratios of the cell and heparin-binding domains, and ultimately modulate the expression of the osteoblast cell phenotype. Approaches similar to the one presented in this work can be used to design materials for hybrid artificial organs and other tissues.

Solute washout experiments for characterizing mass transport in hollow fiber immunoisolation membranes.

The transport characteristics of immunoisolation membranes can have a critical effect on the design of hybrid artificial organs and cell therapies. However, it has been difficult to quantitatively evaluate the desired transport properties of different hollow fiber membranes due to bulk mass transfer limitations in the fiber lumen and annular space. An attractive alternative to existing methodologies is to use the rate of solute removal or "washout" from the annular space during constant flow perfusion through the fiber lumen. Experimental washout curves were obtained for glucose and a 10 kD dextran in two different hollow fiber devices. Data were analyzed using a theoretical model which accounts for convective and diffusive transport in the lumen, membrane, and annular space. The model was in good agreement with the experimental results and provided an accurate measure of the effective membrane diffusion coefficient for both small and large solutes. This approach should prove useful in theoretical analyses of solute transport and performance of hollow fiber artificial organs.

Selected aspects of the microencapsulation of mammalian cells in HEMA-MMA.

Microencapsulation of live mammalian cells is one means of creating hybrid artificial organs, like an artificial pancreas or an artificial liver. In addition to creating and developing the methodologies for enclosing cells within the appropriate semipermeable and biocompatible membranes, novel techniques are needed to assess the various features of the resulting capsules. The small size of a capsule or its heterogeneity can lead to additional complexities that go beyond the problem of examining cell behavior in the presence of biomaterials. These problems are illustrated here by comparison of protein release by microencapsulated HepG2 cells within large and small HEMA-MMA (hydroxyethyl methacrylate-methyl methacrylate) capsules, by assessment of the effect of processing conditions on HEMA-MMA microcapsule permeability to horseradish peroxidase at the individual capsule level, and by a confocal microscopy technique for assessing intracapsule cell viability.

Cytokine synthesis hybrid artificial organs

Cytokine synthesis hybrid artificial organs

Development of a Hybrid Artificial Pancreas with a Dense Polyurethane Membrane

An important element in the evaluation of biomaterials is quantification of the relationships and the sequence of events between blood elements, blood flow, and the foreign surface. We adapted a qualitative two-dimensional 111In-labeled platelet imaging method to a quantitative noninvasive analysis of platelet uptake/release kinetics for infusion catheters in a canine model. Bilateral femoral vein 6 Fr. Groshong catheters (one treated with a hydroxylated siloxane to improve albumin affinity) were monitored at femoral vein sites with a GE 400T gamma camera, interfaced with a Technicare 560 image acquisition computer. The field of view was sufficiently large that all events below the diaphragm were recorded without having to move the camera. Image acquisition time was 2.5 min; images were obtained every 5-15 min for 3 hrs. Continuous recordings were obtained from bilateral ultrasonic velocity probes, attached distal to the catheter implant sites. A 5 ml blood sample was placed in the field to permit calibration of gamma emissions per pixel in terms of labeled platelet density. Signal compensation for near field capillary perfusion was performed. The two-dimensional platelet distribution was computed and displayed. Local, time dependent platelet accumulation on the catheters and adjacent vessel walls was observed. Platelet accumulation proceeded in irregular steps during the implant period. Loss of local platelet deposits was observed. Downstream reattachment of platelet emboli was inferred from simultaneous reductions and increases in local platelet densities at two catheter positions. Platelet attachment was inversely related to vein blood velocity.(ABSTRACT TRUNCATED AT 250 WORDS)

Polydisperse Dextran as a Diffusing Test Solute to Study the Membrane Permeability of Alginate Polylysine Microcapsules

Applications of alginate polylysine (APL) microcapsules in cell culture engineering and hybrid artificial organs require strict control of membrane permeability. For example, hybrid artificial organs must permit the diffusion of smaller molecules including peptides and proteins and the exclusion of leucocytes and immunoglobulins (MW > 150,000). Single molecular weight solutes such as proteins have been used to study the molecular weight cut-off of the membrane. A new approach that uses a heterogenous mixture of dextrans of different molecular weights: -MW 10,000-500,000 as a test solute in diffusion experiments is described. Intra/extra capsular changes in concentration and molecular weight distribution of the heterogenous dextran mixture are monitored and determined by high performance gel chromatography. MW can then be related to diffusional Stokes radius, a more useful parameter when comparing permeability of cell membrane to different test solutes.

Proliferation and Interactions of Several Cell Types Encapsulated Within Dense (Non-Porous) Protein-Permeable Polyurethane Membranes

AbstractProtein-permeable dense (non-porous) urethane membranes have been evaluated forin vitrocell culture, and in vivo cell encapsulation. Polyurethane membranes were designed to exhibit permeability to proteins, gases, and nutrients without the existence of pores. The membranes are non-cytotoxic, angiogenic, and permeable to gases, nutrients, secretagogues and cell products via purely concentration-driven transport. Non-anchorage and anchorage dependent cells were grown encapsulated within the membrane and with the membrane as a growth substrate. Several non-anchorage dependent cell types proliferated within the membrane bothin-vitroandin-vivo. Anchorage-dependent cells were grown on the membranes as a substrate. Encapsulated cells have been maintained in culture for up to six months with nutrients supplied only by the external media. Immuno-isolation has been demonstrated with cells implanted into murine hosts. Explants of membrane encapsulated cells exhibited a high degree of vascularization, with little or no fibrous tissue. The ability to support cell growth and function, and the ability to protect xenogenic cells from immunologic rejection suggest that the membranes would be useful in the construction of hybrid artificial organs, devices for cell transplantation, and substrates for cell and tissue culture.

Two-dimensional orientational response of smooth muscle cells to cyclic stretching.

It was hypothesized that an orientated cellular tissue for incorporation into vital, functioning hybrid artificial organs can be prepared by periodically applying mechanical stresses on a hybrid tissue. Therefore, the effect of cyclic stretching on the two-dimensional (2-D) orientation response of arterial smooth muscle cells (SMCs) was studied. Smooth muscle cells derived from bovine aortas were seeded onto transparent elastomeric membranes made of polyurethane, and subjected to periodical stretching with various amplitudes from 5-20% at frequencies of 15 to 120 RPM for up to 24 hours. Phase-contrast microscopic views of SMCs were time-lapse video recorded. The orientation angle to the direction of stretching (OA) and cellular longitudinal length (CLL) of individual cells were analyzed by a computerized image processor. After several hours, SMCs subjected to the stress exhibited orientation responses perpendicular to the direction of stretching, evidenced by a significant increase in OA. The responses were more rapid under operating conditions with higher amplitudes and frequencies of stretching. Meanwhile, little significant change in CLL was observed. These findings indicate that an applied mechanical stress induces a significant orientation response, without morphologic alteration of SMCs. The mechanically induced orientation response provides a fundamental basis for more structured hybrid organs and tissue engineering.

Hybrid Artificial Cells

In 1965, the use of artificial cells for the microencapsulation of living cells was demonstrated. At that time, this technique was also proposed for use in immunoisolation of endocrine cells, islets, and hepatocytes as hybrid artificial organs. Progress in the biotechnology of cell culture in the last 10 years has allowed this approach to be extensively investigated by an increasing number of centers. The author's recent studies concentrated on microencapsulation of hepatocytes as a model system. Such cells were implanted to increase the survival of galactosamine induced fulminant hepatic failure rats, or to decrease the high bilirubin levels in Gunn rats and xenografts of microencapsulated rat hepatocytes into mice provided immunoisolation. Furthermore, hepatocyte secreted hepatic stimulating factors (mw 110,000 D) accumulating in the microcapsules helps to increase the viability of the hepatocytes. In addition to these studies, a novel, two step cell encapsulation method was developed to improve immunoisolation and biocompatibility. Microencapsulation of microorganisms was also carried out using a model microorganism to convert serum cholesterol to carbon dioxide.

Development of a novel artificial matrix with cell adhesion peptides for cell culture and artificial and hybrid organs.

The authors present a novel artificial matrix with high cell adhesion and growth rate that was produced by chemical fixation of an RGD-containing peptide (Arg-Gly-Asp) on a polyvinyl alcohol (PVA) surface. The logical sequence behind the molecular design of an artificial matrix that mimics natural adhesive proteins, such as fibronectin, is based on the fact that an RGD tripeptidyl amino sequence is the minimal common adhesion site of adhesive proteins. Activation of surface hydroxyl groups by carbonyl diimidazole successfully incorporated GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) onto PVA films. The resultant film surface was found to be bioactive, molecularly recognizing the adhesive receptor of bovine endothelial cells. Thus, the artificial matrix mimics active adhesion sites and serves as a novel artificial matrix that may be useful for cell culture, tissue-compatible implants, and hybrid artificial organs.

The Role of Biotechnology in Bioartificial or Hybrid Artificial Cells and Organs

The Role of Biotechnology in Bioartificial or Hybrid Artificial Cells and Organs