Different regulation of hepatocyte behaviors between natural extracellular matrices and synthetic extracellular matrices by hepatocyte growth factor

The roles of growth factors and extracellular matrices (ECMs) in regulation of hepatocyte behaviors are very important for the establishment of liver-tissue engineering. Especially, collaboration between growth factors and ECMs is a big concern for liver-tissue engineering. In this study, the hepatocyte responses by hepatocyte growth factor (HGF) were compared between natural ECMs and a synthetic galactose-carrying polymer: poly(N-p-vinylbenzyl-4-O-beta-D-galactopyranosyl-D-gluconamide) (PVLA). Hepatocytes underwent proliferation on type I collagen- and fibronectin-coated surfaces in the presence of HGF, whereas hepatocytes formed spheroid on laminin-1-, PVLA-, and poly-L-lysine (PLL)-coated surfaces in the presence of HGF without the activation of proliferation. HGF accelerated ECM deposition, especially laminin-10/11, beneath the hepatocytes cultured on PVLA- and PLL-coated surfaces and the deposited laminin-10/11 activated integrin signaling to collaborate with HGF signaling. Therefore, the deposited ECM molecules should be focused to clear the mechanism of hepatocyte behaviors in the presence of HGF.     

Synthetic sandwich culture of 3D hepatocyte monolayer

The sandwich culture of hepatocytes, between double layers of extra-cellular matrix (ECM), is a well-established in vitro model for re-establishing hepatic polarity and maintaining differentiated functions. Applications of the ECM-based sandwich culture are limited by the mass transfer barriers induced by the top gelled ECM layer, complex molecular composition of ECM with batch-to-batch variation and uncontrollable coating of the ECM double layers. We have addressed these limitations of the ECM-based sandwich culture by developing an 'ECM-free' synthetic sandwich culture, which is constructed by sandwiching a 3D hepatocyte monolayer between a glycine-arginine-glycine-aspatic acid-serine (GRGDS)-modified polyethylene terephthalate (PET) track-etched membrane (top support) and a galactosylated PET film (bottom substratum). The bioactive top support and bottom substratum in the synthetic sandwich culture substituted for the functionalities of the ECM in the ECM-based sandwich culture with further improvement in mass transfer and optimal material properties. The 3D hepatocyte monolayer in the synthetic sandwich culture exhibited a similar process of hepatic polarity formation, better cell-cell interaction and improved differentiated functions over 14-day culture compared to the hepatocytes in collagen sandwich culture. The novel 3D hepatocyte monolayer sandwich culture using bioactive synthetic materials may readily replace the ECM-based sandwich culture for liver tissue engineering applications, such as drug metabolism/toxicity testing and hepatocyte-based bioreactors.     

Some physical and chemical properties of Bhanja virus

Bhanja virus is acid-labile, relatively thermostable, resistant to trypsin and heparin; a complete inactivation was achieved with chloramine B or formaldehyde, while phenol was ineffective, and UV radiation only partially effective.     

Tailoring the pore architecture in 3-D alginate scaffolds by controlling the freezing regime during fabrication

The pore architecture in 3-D polymeric scaffoldings plays a critical role in tissue engineering as it provides the framework for the seeded cells to organize into a functioning tissue. In the present paper, we investigate the effect of freezing regime on the pore microstructure in 3-D alginate scaffolds, fabricated by the freeze-dry method. The scaffolds have shown isotropic pore structure, when the calcium crosslinked alginate solutions were slowly frozen at -20 degrees C, in a nearly homogenous cold atmosphere; the pores were spherical and interconnected. In contrast, when the cooling process was performed in liquid nitrogen or oil bath, where a temperature gradient was formed along the freezing solution, two main regions of pore structure were noted; at the interface with the cooling medium, small spherical pores were seen and above them a region with elongated pores. The different pore shape affected the compressibility of the scaffolds, while it had no effect on albumin diffusion. Rat hepatocytes seeded within the scaffolds were arranged according to the their pore shape. In scaffolds with elongated pores, the cells were lining along the pores, thus forming lines of interacting cells. In the scaffolds with the isotropic spherical pores, the hepatocytes clustered into spheroid-like aggregates. Thus, it appears that pore shape can modulate hepatocyte morphogenesis.     

3-D physical models of amitosis (cytokinesis)

Based on Newton's laws, extended Coulomb's law and published biological data, we develop our 3-D physical models of natural and normal amitosis (cytokinesis), for prokaryotes (bacterial cells) in M phase. We propose following hypotheses: Chromosome rings exclusion: No normally and naturally replicated chromosome rings (RCR) can occupy the same prokaryote, a bacterial cell. The RCR produce spontaneous and strong electromagnetic fields (EMF), that can be alternated environmentally, in protoplasm and cortex. The EMF is approximately a repulsive quasi-static electric (slowly variant and mostly electric) field (EF). The EF forces between the RCR are strong enough, and orderly accumulate contractile proteins that divide the procaryotes in the cell cortex of division plane or directly split the cell compartment envelope longitudinally. The radial component of the EF forces could also make furrows or cleavages of procaryotes. The EF distribution controls the protoplasm partition and completes the amitosis (cytokinesis). After the cytokinesis, the spontaneous and strong EF disappear because the net charge accumulation becomes weak, in the protoplasm. The exclusion is because the two sets of informative objects (RCR) have identical DNA codes information and they are electro magnetically identical, therefore they repulse from each other. We also compare divisions among eukaryotes, prokaryotes, mitochondria and chloroplasts and propose our hypothesis: The principles of our models are applied to divisions of mitochondria and chloroplasts of eucaryotes too because these division mechanisms are closer than others in a view of physics. Though we develop our model using 1 division plane (i.e., 1 cell is divided into 2 cells) as an example, the principle of our model is applied to the cases with multiple division planes (i.e., 1 cell is divided into multiple cells) too.     

Study on the physical properties of tissue-engineered blood vessels made by chemical cross-linking and polymer-tissue cross-linking

In this study, we attempted to chemically cross-link decellularized blood vessel tissue and to perform cross-linking with a polymer in order to control its stability and functionalization. For this purpose, we cross-linked tissue by intrahelical, interhelical, and intermolecular cross-linking between the polymer and the collagen helix, which is a component of the native tissue. The intrahelically cross-linked tissue showed weaker stability against heat and degradation caused by collagenase compared to the interhelically cross-linked tissue. The tissue intermolecularly cross-linked with polymer showed the highest stability against heat and degradation caused by collagenase. The mechanical strength test showed that the Young’s moduli were different for the intra/interhelically and intermolecularly cross-linked tissues, with the latter being stiffer. This is thought to be because the cross-linked polymer functions in the same way as elastin, whereas simple collagen cross-linking provides a supportive matrix that holds the collagen and elastin together.     

Relationship between physical and chemical properties of aluminum-containing adjuvants and immunopotentiation

Aluminum-containing adjuvants are an important component of many vaccines because they safely potentiate the immune response. The structure and properties of aluminum hydroxide adjuvant, aluminum phosphate adjuvant and alum-precipitated adjuvants are presented in this review. The major antigen adsorption mechanisms, electrostatic attraction and ligand exchange, are related to the adjuvant structure. The manner by which aluminum-containing adjuvants potentiate the immune response is related to the structure, properties of the adjuvant and adsorption mechanism. Immunopotentiation occurs through the following sequential steps: inflammation and recruitment of antigen-presenting cells, retention of antigen at the injection site, uptake of antigen, dendritic cell maturation, T-cell activation and T-cell differentiation.     

Development of PDMS microbioreactor with well-defined and homogenous culture environment for chondrocyte 3-D culture

Perfusion cell culture is believed to provide a stable culture environment due to the continuous supply of nutrients and removal of waste. However, the culture scales used in most cases were large, where the culture conditions can not be regarded as homogenous because of chemical gradients. To improve this, the concept of miniaturization is applied to 3-D cell culture. In this study, a simple perfusion microbioreactor was developed based on mass transport simulation to find out the reasonable culture scales with relatively lower chemical gradients. Besides, PDMS surface was treated with surfactant solution to reduce non-specific serum protein adsorption, which keeps the culture conditions steady. Chondrocyte 3-D culture using the proposed microbioreactors was compared with similar perfusion culture with a larger culture scale. Results showed that surfactant-treated PDMS surface could reduce serum protein adsorption by 85% over the native one. Also, microbioreactors were proved to provide a stable culture environment (e.g. pH) over the culture period. Cell culture scale of 200 μm thick culture construct was justified to have relatively lower chemical gradients than the larger scale perfusion culture. As a whole, the proposed culture system is capable of providing a well-defined and homogenous culture environment.     

磷酸钙陶瓷的理化性能与宿主组织的关系

磷酸钙陶瓷如磷酸三钙 (TCP)和羟基磷灰石 (HA)是常用人工骨替代材料。这些材料具有不同的理化性能 ,因而能与宿主组织发生不同的反应。多孔性、骨传导性、生物相容性均是研制人工骨替代材料时要考虑的重要因素。本文介绍了磷酸钙陶瓷的理化性能与宿主组织之间的关系     

Tissue specific synthetic ECM hydrogels for 3-D in vitro maintenance of hepatocyte function

Despite recent advances in biomaterial science, there is yet no culture system that supports long-term culture expansion of human adult hepatocytes, while preserving continued function. Previous studies suggested that acellular liver extracellular matrix (ECM), employed as a substrate, improved proliferation and function of liver cells. Here we investigated whether extracts prepared from acellular liver ECM (liver ECM extract, LEE), or from whole (fresh) liver tissue (liver tissue extract, LTE), could be combined with collagen Type I, hyaluronic acid (HA), or heparin-conjugated HA (HP) hydrogels to enhance survival and functional output of primary human hepatocytes. The liver-specific semi-synthetic ECMs (sECMs) were prepared by incorporating LEE or LTE into the gel matrices. Subsequently, primary human hepatocytes were maintained in sandwich-style hydrogel cultures for 4 weeks. Progressive increase in hepatocyte metabolism was observed in all HA and HP groups. Hepatocytes cultured in HA and HP hydrogels containing LEE or LTE synthesized and secreted steady levels of albumin and urea and sustained cytochrome p450-dependent drug metabolism of ethoxycoumarin. Collectively, these results indicate that customized HA hydrogels with liver-specific ECM components may be an efficient method for expansion human hepatocytes in vitro for cell therapy and drug and toxicology screening purposes.     

A novel 3-D model for cell culture and tissue engineering

A novel method of making microcapsules in a macrocapsule is demonstrated as a 3-D culture system in this article. Mouse embryonic stem (mES) cells as model cells were used in the 3-D culture space, and the cell viability and histological observation were conducted. Furthermore, Oct4 gene expression was evaluated for the undifferentiated status of mES cells in this 3-D model. The results showed that mES cells can grow in this 3-D model and retain their normal viability and morphology. This 3-D model allows mES cells to stay in the undifferentiated state better than 2-D culture systems. This work demonstrates a new 3-D tissue model which can provide an in vivo like microenvironment for non-differentiated mES cells with good immunoisolation. This approach may bridge the gap between traditional 2-D cell culture and animal models.     

A Bence Jones cryoglobulin: chemical, physical and immunological properties

A Bence Jones protein with cryoglobulin properties has been isolated from the urine of a myeloma patient. The Bence Jones cryoglobulin was of type K and had a sedimentation coefficient of 3·5S at a concentration of 5 mg/ml. The concentration dependence of cryoglobulin formation suggested that it was due to reversible association. This was confirmed by ultracentrifugal analyses which demonstrated the formation of aggregates with a sedimentation coefficient of approximately 50–100S and higher. Quantitative amino acid analyses revealed that the Bence Jones cryoglobulin was completely devoid of methionine. On starch gel electrophoresis, three to four protein bands could be detected. They were all devoid of methionine and there was no significant difference in their content of other amino acids. On the basis of amino acid and ultracentrifugal analyses the presence of several components on starch gel electrophoresis is consistent with the formation of aggregates by a single molecular species.     

Gastric parietal cell autoantigen: physical, chemical and biological properties

The properties of the gastric parietal cell autoantigen have been further investigated using mucosal subcellular fractions and gastric sections. Sucrose density layer ultracentrifugation of a microsomal preparation from bovine abomasum fundus (gastric) mucosa allowed the separation of an antigenic smooth membrane fraction, providing further evidence that the antigen is associated with smooth cytoplasmic membranes. Both hydrophobic and polar bonds appear to be concerned with maintenance of antigenic structure. Protein and lipid are essential components of the antigen. Lipid extracted with chloroform–methanol was inactive. The lipid-free protein retained slight activity which was increased on recombination with the lipid. The results suggest that the antigen is a lipoprotein. Enzymatic and chemical treatments showed that RNA and carbohydrate are not concerned in the antigenicity, which was also not dependent upon thiol groups, disulphide bonds or metal ions. Attempts to subfractionate an antigenic 6 M urea extract of bovine gastric microsomes by gel filtration and electrophoresis led to recovery of antigenic material without further resolution. An immunoadsorption–elution experiment gave promise of this being a useful method for purification of the antigen.     

Control of hepatocyte function on collagen foams: sizing matrix pores toward selective induction of 2-D and 3-D cellular morphogenesis

While microporous biopolymer matrices are being widely tested as cell culture substrates in hepatic tissue engineering, the microstructural basis for their control of cell differentiation is not well understood. In this paper, we studied the role of void size of collagen foams in directing the induction of liver-specific differentiated morphology and secretory activities of cultured rat hepatocytes. Hepatocytes cultured on collagen foams with subcellular sized pore diameters of 10 microm assumed a compact, cuboidal cell morphology, rapidly achieving monolayer coverage, and secreted albumin at the rate of 40 +/- 8 pg/cell/d. Increasing the pore size to 18 microm elicited a distinctly spread cellular phenotype, with discontinuous surface coverage, and albumin secretion rates declined precipitiously to 3.6 +/- 0.8 pg/cell/d. However, when collagen foams with an even higher average void size of 82 microm were used, hepatocytes exhibited high degree of spreading within an extensive three-dimensional cellular network, and exhibited high albumin secretory activity (26 +/- 0.6 pg/cell/d). The effect of void geometry on cellular ultrastructral polarity was further analyzed for the three void size configurations employed. The distribution of the cell-cell adhesion protein, E-cadherin, was primarily restricted to cell-cell contacts on the 10 microm foams, but was found to be depolarized to all membrane regions in cells cultured on the 18 and 82 microm foams. Vinculin-enriched focal adhesions were found to be peripherally clustered on cells cultured on 10 microm foams, but were found to redistribute to the entire ventral surface of cells cultured on the 18 and 82 microm foams. Overall, we demonstrate the significance of designing pore sizes of highly adhesive substrates like collagen toward selective cell morphogenesis in 2-D or 3-D. Subcellular and supercellular ranges of pore size promote hepatocellular differentiation by limiting 2-D cell spreading or effecting 3-D intercellular contacts, while intermediate range of pore sizes repress differentiation by promoting 2-D cell spreading.     

Preparation and culture of hepatocyte on gelatin microcarriers

Porous gelatin microcarriers having a diameter of 80-100 microm were prepared by the suspension method using toluene as the oil phase. Rat hepatocytes were cultured on gelatin and cytodexIII microcarriers. The cells retained its spherical shape, which is similar in vivo, and showed no morphological changes to the flat state. Hepatocyte aggregates on microcarriers maintained higher metabolic functions than monolayer cells. Pore size of microcarrier plays an important role in the attachment and metabolic function of cells in culture. Phase-contrast micrograph and cell activity showed that hepatocytes cultures on gelatin microcarrier of <1 microm pore size is better than that of 5-10 microm.     

DLC coatings: effects of physical and chemical properties on biological response

Recent trials on diamond-like carbon (DLC) coated medical devices have indicated promise for blood interfacing applications. The literature is sparse regarding structural and compositional effects of DLC on cellular response. An important goal in optimizing blood-interfacing implants is minimal macrophage attachment, and maximal albumin:fibrinogen adsorption ratio. DLC coatings deposited by PACVD and FAD, were analysed with respect to sp3 content (EELS), hydrogen content (ERDA), surface composition (XPS), surface roughness (AFM), surface energy, albumin:fibrinogen adsorption ratio, and macrophage viability and attachment. We found that increasing surface roughness and surface energy enhanced the macrophage viability and the albumin:fibrinogen adsorption ratio. We also found that the higher the hydrogen content for a-C:Hs deposited by PACVD, the lower the albumin:fibrinogen adsorption ratio, and macrophage attachment. This suggests that hydrogen content may be an important factor for influencing the biological response of DLC surfaces. Macrophage cells spread well on all DLC surfaces, and the surface results indicated the non-toxic nature of the surfaces on the cells at the time points tested.     

Primary hepatocyte survival on non-integrin-recognizable matrices without the activation of Akt signaling

The suppression of the detachment-induced cell death (anoikis) by the interaction between the cells and extracellular matrix (ECM) is necessary for the application of liver tissue engineering because the disruption of interaction with ECM leads hepatocytes to anoikis. It has been considered, in general, that integrin signal plays an important role in the hepatocyte survival although hepatocytes survive on some types of non-integrin-recognizable matrices, such as poly(N-p-vinylbenzyl-4-O-beta-D-galactopyranosyl-D-gluconamide) (PVLA) and poly-L-lysine (PLL) for several days without the serum. Anoikis was suppressed in the non-adherent culture of hepatocytes isolated from gld/gld mouse, indicating that Fas signal induces hepatocyte anoikis. Fas production is decreased in the adherent culture of hepatocytes on both integrin- and non-integrin-recognizable matrices. Akt activation was hardly observed in the adherent culture of hepatocytes on non-integrin-recognizable matrices whereas the activation occurred in the adherent culture on integrin-recognizable matrices. In the adherent culture of hepatocytes on non-integrin-recognizable matrices, Akt does not contribute to the hepatocyte survival. To prolong the viability of hepatocytes in the adherent culture on PVLA matrix on which hepatocytes maintain their functions for longer period than those on PLL matrix, it might be a good approach to activate Akt signaling pathway.