Development and Characterization of an Acellular Porcine Small Intestine Submucosa Scaffold for Use in Corneal Epithelium Tissue Engineering.

Purpose: To produce an acellular small intestine submucosa (SIS) that would be a suitable scaffold for corneal epithelium tissue engineering.Methods: The SIS was decellularized by immersion in 0.1% (wt/vol) sodium dodecyl sulfate (SDS). The efficacy of acellularization was confirmed by histological observation and DNA quantification. The mechanical properties were evaluated by uniaxial tensile testing. ELISA was performed to assess the growth factor contents. The cytotoxicity of SIS scaffolds and extracts to rabbit corneal epithelial cells was determined by CCK-8 assay. We also investigated the inflammatory reaction of SIS implanted subcutaneously in a rat. The biocompatibility was studied by rabbit interlamellar corneal transplantation and reseeding assay with cornea-derived cells. Immunofluorescent staining was used to detect the expression of CK3, ZO-1 and K13.Results: Histological analyses showed that complete cell removal was achieved, and the DNA quantity, which reflects the presence of cellular materials, was significantly diminished in acellular SIS. Collagen fibers were properly preserved and appeared in an orderly fashion. The tissue structure, the mechanical properties and the growth factor contents within the acellular SIS were well retained. The CCK8 assay demonstrated that the acellular SIS scaffolds and extracts had no cytotoxicity to rabbit corneal epithelial cells. There was no sign that an immune reaction occurred with acellular SIS implanted subcutaneously in a rat. In fact, in vivo implantation to rabbit interlamellar stromal pockets showed good biocompatibility. We also observed that clusters of rabbit corneal epithelial cells were growing well on the surface of the SIS in vitro and the distinctive CK3, ZO-1 for corneal epithelial cells was detected.Conclusions: The decellularized SIS retained the major structural components. The matrix is biocompatible with cornea-derived cells and might be a suitable scaffold for corneal epithelium tissue engineering.

Mechanics and dynamics of reconstituted cytoskeletal systems.

The intracellular cytoskeleton is an active dynamic network of filaments and associated binding proteins that control key cellular properties, such as cell shape and mechanics. Due to the inherent complexity of the cell, reconstituted model systems have been successfully employed to gain an understanding of the fundamental physics governing cytoskeletal processes. Here, we review recent advances and key aspects of these reconstituted systems. We focus on the importance of assembly kinetics and dynamic arrest in determining network mechanics, and highlight novel emergent behavior occurring through interactions between cytoskeletal components in more complex networks incorporating multiple biopolymers and molecular motors.

Structural and biomechanical characterizations of porcine myocardial extracellular matrix.

Extracellular matrix (ECM) of myocardium plays an important role to maintain a multilayered helical architecture of cardiomyocytes. In this study, we have characterized the structural and biomechanical properties of porcine myocardial ECM. Fresh myocardium were decellularized in a rotating bioreactor using 0.1 % sodium dodecyl sulfate solution. Masson's trichrome staining and SEM demonstrated the removal of cells and preservation of the interconnected 3D cardiomyocyte lacunae. Movat's pentachrome staining showed the preservation of cardiac elastin ultrastructure and vascular elastin distribution/alignment. DNA assay result confirmed a 98.59 % reduction in DNA content; the acellular myocardial scaffolds were found completely lack of staining for the porcine α-Gal antigen; and the accelerating enzymatic degradation assessment showed a constant degradation rate. Tensile and shear properties of the acellular myocardial scaffolds were also evaluated. Our observations showed that the acellular myocardial ECM possessed important traits of biodegradable scaffolds, indicating the potentials in cardiac regeneration and whole heart tissue engineering.

Reversible membrane permeabilization of mammalian cells treated with digitonin and its use for inducing nuclear reprogramming by Xenopus egg extracts.

Plasma membranes can be reversibly permeabilized by Streptolysin O. The permeabilized cells can be reprogrammed and partially dedifferentiated in the cell-free system from egg extracts. However, the permeabilizing activity of Streptolysin O is not stable, and therefore it is difficult to control its activity. An alternative method for reversible permeabilization is useful for establishing a cell-free system. Here, we used a nonionic detergent, digitonin, for permeabilization. A low concentration of digitonin induced reversible permeabilization of the plasma membrane in bovine, mouse, and porcine somatic cells. The permeabilized cells were treated with Xenopus laevis egg extracts. The treated cells showed exchange of nuclear proteins from extracts such as incorporation of Xenopus-specific histone B4 and Lamin LIII into nuclei. After resealing of the membrane, the cells showed upregulation of OCT4, SOX2, and NANOG expression. Our results suggest that reversible permeabilization with digitonin can be used to induce nuclear reprogramming and to activate pluripotent genes by a cell-free system.

Docking and fusion of synaptic vesicles in cell-free model system of exocytosis.

The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro. The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca(2+) concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca(2+)-independent step, termed docking and followed by fusion step that is triggered by Ca(2+). The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed.

Molecular mechanism of mitotic Golgi disassembly and reassembly revealed by a defined reconstitution assay.

In mammalian cells, flat Golgi cisternae closely arrange together to form stacks. During mitosis, the stacked structure undergoes a continuous fragmentation process. The generated mitotic Golgi fragments are distributed into the daughter cells, where they are reassembled into new Golgi stacks. In this study, an in vitro assay has been developed using purified proteins and Golgi membranes to reconstitute the Golgi disassembly and reassembly processes. This technique provides a useful tool to delineate the mechanisms underlying the morphological change. There are two processes during Golgi disassembly: unstacking and vesiculation. Unstacking is mediated by two mitotic kinases, cdc2 and plk, which phosphorylate the Golgi stacking protein GRASP65 and thus disrupt the oligomer of this protein. Vesiculation is mediated by the COPI budding machinery ARF1 and the coatomer complex. When treated with a combination of purified kinases, ARF1 and coatomer, the Golgi membranes were completely fragmented into vesicles. After mitosis, there are also two processes in Golgi reassembly: formation of single cisternae by membrane fusion, and restacking. Cisternal membrane fusion requires two AAA ATPases, p97 and NSF (N-ethylmaleimide-sensitive fusion protein), each of which functions together with specific adaptor proteins. Restacking of the newly formed Golgi cisternae requires dephosphorylation of Golgi stacking proteins by the protein phosphatase PP2A. This systematic study revealed the minimal machinery that controls the mitotic Golgi disassembly and reassembly processes.

Fabrication of a PLGA-collagen peripheral nerve scaffold and investigation of its sustained release property in vitro.

This study deals with the fabrication of a peripheral nerve scaffold prepared with poly (lactic acid-co-glycolic acid) [PLGA] and acellularized pigskin collagen micro particles and the investigation of its sustained release property in vitro. We took bovine serum albumin [BSA] as model drug to investigate the sustained-release property of the scaffold in vitro. The results showed the scaffold could release BSA steadily with a rate of 6.6 ng/d (r=0.994) or so. In a 1-month test period, the accumulative release ratio of BSA from the scaffold was up to 43%, and the shape of the scaffold was still originally well kept. In addition, the scaffold outcome non-immunogenicity, good cell adhesion and biodegradability. The results indicated a scaffold constructed by this technique would be a potential implanting support with prolonged sustained release function, such as for the use of nerve scaffold.

Lysosomes: fusion and function.

Lysosomes are dynamic organelles that receive and degrade macromolecules from the secretory, endocytic, autophagic and phagocytic membrane-trafficking pathways. Live-cell imaging has shown that fusion with lysosomes occurs by both transient and full fusion events, and yeast genetics and mammalian cell-free systems have identified much of the protein machinery that coordinates these fusion events. Many pathogens that hijack the endocytic pathways to enter cells have evolved mechanisms to avoid being degraded by the lysosome. However, the function of lysosomes is not restricted to protein degradation: they also fuse with the plasma membrane during cell injury, as well as having more specialized secretory functions in some cell types.

Generation of cell-free extracts of Xenopus eggs and demembranated sperm chromatin for the assembly and isolation of in vitro-formed nuclei for Western blotting and scanning electron microscopy (SEM).

This protocol details methods for the generation of cell-free extracts and DNA templates from the eggs and sperm chromatin, respectively, of the clawed toad Xenopus laevis. We have used this system with scanning electron microscopy (SEM), as detailed herein, to analyze the biochemical requirements and structural pathways for the biogenesis of eukaryotic nuclear envelopes (NEs) and nuclear pore complexes (NPCs). This protocol requires access to female frogs, which are induced to lay eggs, and a male frog, which is killed for preparation of the sperm chromatin. Egg extracts should be prepared in 1 d and can be stored for many months at -80 degrees C. Demembranated sperm chromatin should take only approximately 2-3 h to prepare and can be stored at -80 degrees C almost indefinitely. The time required for assembly of structurally and functionally competent nuclei in vitro depends largely on the quality of the cell-free extracts and, therefore, must be determined for each extract preparation.

Neuronal expression of the chondroitin sulfate proteoglycans receptor-type protein-tyrosine phosphatase beta and phosphacan.

Receptor-type protein-tyrosine phosphatase beta (RPTPbeta) and its spliced variant phosphacan are major components of chondroitin sulfate proteoglycans in the CNS. In this study, expression and localization of RPTPbeta and phosphacan were examined in developing neurons by immunological analyses using 6B4, 3F8, and anti-PTP antibodies and reverse transcription-polymerase chain reaction (RT-PCR). Light microscopic immunohistochemistry showed that 6B4 RPTPbeta/phosphacan immunoreactivity was observed around neurons in the cortical plate. Further ultrastructural observation showed that 6B4 RPTPbeta/phosphacan immunoreactivity was observed mainly at the membrane of migrating neurons and radial glia. Immunocytochemical analysis revealed that RPTPbeta immunoreactivity was observed in cultured cerebral, hippocampal, and cerebellar neurons in addition to type-1 and type-2 astrocytes. Western analysis further demonstrated that the shorter receptor form of RPTPbeta (sRPTPbeta) was detected from cell lysate of cortical and hippocampal neurons using 6B4 and anti-PTP antibodies, while sRPTPbeta of cerebellar neurons and type-1 astrocytes was recognized only by anti-PTP antibody. Phosphacan was detected from neuronal culture supernatants of cortical, hippocampal, and cerebellar neurons, but not from type-1 astrocytes using 6B4 and 3F8 antibodies. RT-PCR analysis demonstrated the prominent expression of sRPTPbeta and phosphacan mRNAs in cortical neurons, and that of sRPTPbeta mRNA in type-1 astrocytes. During culture development of cortical neurons, the immunoreactivity of 6B4 sRPTPbeta was observed entirely on the neuronal surface including somata, dendrites, axons, and growth cones at earlier stages of cortical neuronal culture such as stages 2 and 3, while, after longer culture, 6B4 sRPTPbeta immunoreactivity in stages 4 and 5 neurons was detected at dendrites and somata and disappeared from axons, and was not observed over axonal terminals and postsynaptic spines. These results demonstrate that neurons are able to express sRPTPbeta on their cellular surface and to secrete phosphacan, and neuronal expression of sRPTPbeta may modulate neuronal differentiation including neuritogenesis and synaptogenesis.

Construction of varying porous structures in acellular bovine pericardia as a tissue-engineering extracellular matrix.

In the study, a cell extraction process was used to remove the cellular components from bovine pericardia. Varying pore sizes and porosities of the acellular tissues were then created using acetic acid and collagenase and subsequently fixed with genipin. Biochemical analyses found that these acellular tissues with distinct porous structures consisted primarily of insoluble collagen, elastin, and tightly bound glycosaminoglycans. The thermal stability, mechanical properties, and capability against enzymatic degradation of the bovine pericardial tissue remained unaltered after cell extraction. However, following further treatment with acetic acid and collagenase, the thermal stability and capability against enzymatic degradation of the acellular tissues declined. The porous structures of the implanted samples seem to determine whether successful microvessel-ingrowth takes place. The acetic-acid- and collagenase-treated tissues, due to their high pore size and porosity, showed a large number of microvessels infiltrating into the interstices of the implanted samples. In contrast, a low density of microvessels was observed infiltrating into the acellular tissue and penetration of microvessels into the cellular tissue was never encountered.

Vascular patches tissue-engineered with autologous bone marrow-derived cells and decellularized tissue matrices.

Synthetic polymer vascular patches used in cardiovascular surgery have shortcomings such as thrombosis, intimal hyperplasia, calcification, infection, and no growth potential. Tissue-engineered vascular patches using autologous vascular cells may solve these problems. In this study, we developed a tissue-engineered vascular patch using autologous bone marrow-derived cells (BMCs) and decellularized tissue matrices. Vascular smooth muscle cells and endothelial cells were differentiated from bone marrow mononuclear cells in vitro. Tissue-engineered vascular patches were fabricated by seeding these cells onto decellularized canine inferior vena cava matrices and implanted into the inferior vena cava of dogs. Three weeks after implantation, the tissue-engineered vascular patches were patent with no sign of thrombus formation. Histological, immunohistochemical, and electron microscopic analyses of the vascular patches retrieved 3 weeks after implantation revealed regeneration of endothelium and smooth muscle and the presence of collagen and elastin. BMCs labeled with a fluorescent dye prior to implantation were detected in the retrieved vascular patches, indicating that the BMCs survived after implantation and contributed to the vascular tissue regeneration. This study demonstrates that vascular patches can be tissue-engineered with autologous BMCs and decellularized tissue matrices.

Advances in the analysis of single mitochondria.

Mitochondria are both morphologically and functionally diverse, and this variety is thought to have important biological ramifications. The development of methods to probe the properties of individual mitochondria is therefore of utmost importance. Recent advances have been made using in situ microscopy techniques and methods to investigate isolated mitochondria, including flow cytometry, capillary electrophoresis, patch-clamping and optical trapping. Such techniques have been used to study metabolism, mitochondrial calcium homeostasis, mitochondrial membrane potential, apoptosis, and other properties.

TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/zeta complex.

We show in this study that human T cells purified from peripheral blood, T cell clones, and Jurkat T cells release microvesicles in the culture medium. These microvesicles have a diameter of 50-100 nm, are delimited by a lipidic bilayer membrane, and bear TCR beta, CD3epsilon, and zeta. This microvesicle production is regulated because it is highly increased upon TCR activation, whereas another mitogenic signal, such as PMA and ionomycin, does not induce any release. T cell-derived microvesicles also contain the tetraspan protein CD63, suggesting that they originate from endocytic compartments. They contain adhesion molecules such as CD2 and LFA-1, MHC class I and class II, and the chemokine receptor CXCR4. These transmembrane proteins are selectively sorted in microvesicles because CD28 and CD45, which are highly expressed at the plasma membrane, are not found. The presence of phosphorylated zeta in these microvesicles suggests that the CD3/TCR found in the microvesicles come from the pool of complexes that have been activated. Proteins of the transduction machinery, tyrosine kinases of the Src family, and c-Cbl are also observed in the T cell-derived microvesicles. Our data demonstrate that T lymphocytes produce, upon TCR triggering, vesicles whose morphology and phenotype are reminiscent of vesicles of endocytic origin produced by many cell types and called exosomes. Although the exact content of T cell-derived exosomes remains to be determined, we suggest that the presence of TCR/CD3 at their surface makes them powerful vehicles to specifically deliver signals to cells bearing the right combination of peptide/MHC complexes.

P67-phox-mediated NADPH oxidase assembly: imaging of cytochrome b558 liposomes by atomic force microscopy.

NADPH oxidase activity depends on the assembly of the cytosolic activating factors, p67-phox, p47-phox, p40-phox, and Rac with cytochrome b(558). The transition from an inactive to an active oxidase complex induces the transfer of electrons from NADPH to oxygen through cytochrome b(558). The assembly of oxidase complex was studied in vitro after reconstitution in a heterologous cell-free assay by using true noncontact mode atomic force microscopy. Cytochrome b(558) was purified from neutrophils and Epstein-Barr virus-immortalized B lymphocytes and incorporated into liposomes. The effect of protein glycosylation on liposome size and oxidase activity was investigated. The liposomes containing the native hemoprotein purified from neutrophils had a diameter of 146 nm, whereas after deglycosylation, the diameter was reduced to 68 nm, although oxidase activity was similar in both cases. Native cytochrome b(558) was used after purification in reconstitution experiments to investigate the topography of NADPH oxidase once it was assembled. For the first time, atomic force microscopy illustrated conformational changes of cytochrome b(558) during the transition from the inactive to the active state of oxidase; height measurements allow the determination of a size of 4 nm for the assembled complex. In the processes that were studied, p67-phox displayed a critical function; it was shown to be involved in both assembly and activation of oxidase complex while p47-phox proceeded as a positive effector and increased the affinity of p67-phox with cytochrome b(558), and p40-phox stabilizes the resting state. The results suggest that although an oligomeric structure of oxidase machinery has not been demonstrated, allosteric regulation mechanisms may be proposed.

Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles.

Activation-induced cell death is a process by which overactivated T cells are eliminated, thus preventing potential autoimmune attacks. Two known mediators of activation-induced cell death are Fas(CD95) ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL). We show here that upon mitogenic stimulation, bioactive FasL and APO2L are released from the T cell leukemia Jurkat and from normal human T cell blasts as intact, nonproteolyzed proteins associated with a particulate, ultracentrifugable fraction. We have characterized this fraction as microvesicles of 100-200 nm in diameter. These microvesicles are released from Jurkat and T cell blasts shortly (

Cell-free analysis of Golgi apparatus membrane traffic in rat liver.

Cell-free systems for the analysis of Golgi apparatus membrane traffic rely either on highly purified cell fractions or analysis by specific trafficking markers or both. Our work has employed a cell-free transfer system from rat liver based on purified fractions. Transfer of any constituent present in the donor fraction that can be labeled (protein, phospholipid, neutral lipid, sterol, or glycoconjugate) may be investigated in a manner not requiring a processing assay. Transition vesicles were purified and Golgi apparatus cisternae were subfractionated by means of preparative free-flow electrophoresis. Using these transition vesicles and Golgi apparatus subfractions, transfer between transitional endoplasmic reticulum and cis Golgi apparatus was investigated and the process subdivided into vesicle formation and vesicle fusion steps. In liver, vesicle formation exhibited both ATP-independent and ATP-dependent components whereas vesicle fusion was ATP-independent. The ATP-dependent component of transfer was donor and acceptor specific and appeared to be largely unidirectional, i.e., ATP-dependent retrograde (cis Golgi apparatus to transitional endoplasmic reticulum) traffic was not observed. ATP-dependent transfer in the liver system and coatomer-driven ATP-independent transfer in more refined yeast and cultured cell systems are compared and discussed in regard to the liver system. A model mechanism developed for ATP-dependent budding is proposed where a retinol-stimulated and brefeldin A-inhibited NADH protein disulfide oxidoreductase (NADH oxidase) with protein disulfide-thiol interchange activity and an ATP-requiring protein capable of driving physical membrane displacement are involved. It has been suggested that this mechanism drives both the cell enlargement and the vesicle budding that may be associated with the dynamic flow of membranes along the endoplasmic reticulum-vesicle-Golgi apparatus-plasma membrane pathway.

[The nuclear scaffold structures and plasmid DNA replication in the cells of higher eukaryotes]. / Skeletnye struktury iadra i replikatsiia plazmidnoi DNK v kletkakh vysshikh éukariot.

Incubation of purified plasmid DNA with nuclear matrix extracts enhances efficacy of DNA replication in Xenopus egg extracts. This enhancement correlates with the accelerated rate of pseudo-nuclei formation.

Non-machinery-based system for cell-free, concentrated autogenous ascitic fluid reinfusion.

A non-machinery-based system for the reinfusion of ascitic fluid was developed and assessed. In fundamental studies utilizing bovine serum, this procedure proved economical, quick and useful. The most suitable filter was PS-R (#405-2). Bovine serum with a protein concentration below 3.0 g/dl was treated using this system. Samples containing blood (prepared to 0.5% hematocrit) were also treated, but the treatment time required was double that of serum with the same protein concentration. In both cases the protein recovery ratios were about 90%. We conducted clinical studies on 62 occasions (machinery-based system; 31 times, non-machinery-based system; 31 times) on 19 cases of ascites refractory to treatment with various drugs including diuretics. Clarification of the differences between the non-machinery-based system, indicated the former to be superior. This new procedure is easier because of its use of no machinery, and the high protein recovery ratio proved its usefulness.

[The roles of two kinds of membrane vesicles in the formation of annulate lamellae and nuclear envelopes in a cell-free system from Xenopus egg extracts].

The eggs of Xenopus laevis were activated to complete their second meiosis and enter interphase by calcium ionophore A 23187. Then they were centrifuged at 10,000 g to get activated Xenopus egg extracts. Chromatin structure could form in such an extracts after exogenous Lambda DNA was added, and nuclear envelopes (NEs) could assemble around the chromatin. Furthermore, annulate lamellae (AL), a NE-like membrane structure, could form in chromatin-free regions simultaneously. After large amounts of observation and analysis at ultrastructural level, we propose that both AL and NEs assemble in the extracts by the fusion of membrane vesicles. There were two kinds of membrane vesicles in Xenopus egg extracts, small rough vesicles with diameters of 200 nm and large smooth vesicles. They had many differences in morphology, size and membrane structure. However, they both participate in the formation of AL and NEs, 200 nm membrane vesicles first fused each other to become parallel double membrane cisternae, nuclear pore complexes assembled in the double membranes in the mean time. The fusion of large smooth vesicles with the double membrane cirternae led to the growth of AL and the maturity of NEs. During the formation of NEs, large smooth vesicles were easily observed to link two neighboring double membrane fragments by fusing with them, so that integrated nuclear envelope could form. 200 nm small rough vesicles could attach to the surface of chromatin to start the assembly of nuclear envelopes, while others could fuse each other at chromatin-free regions and these fusion resulted in the formation of annulate lamellae.