Hepatocytic Cells Form Bile Duct-like Structures within a Three-Dimensional Collagen Gel Matrix

It has been suggested that hepatocytes have the ability to form bile ductal structures during normal development and in various pathological conditions of the liver. In the present study, we attempted to establish anin vitromodel of ductal morphogenesis of hepatocytic cells by combining an aggregate culture and a type I collagen gel culture. When spheroidal aggregates of rat or mouse primary hepatocytes were embedded within the collagen gel matrix and then cultured with a medium containing a fibroblast-conditioned medium, the aggregates extended many dendritic processes composed of a trabecular arrangement of cells. Dendritic morphogenesis was also seen in embedded aggregates of immortal liver epithelial cell lines, which spontaneously emerged during long-term cultures of mouse primary hepatocytes. A similar morphogenesis was induced by the presence of insulin in the medium. Although epidermal growth factor (EGF) and hepatocyte growth factor (HGF) showed only a small effect on the morphogenesis of most of the hepatocytic cells when used alone, these factors, especially EGF, enhanced the morphogenetic effect of insulin. Electron microscopical observations revealed luminal structures lined by microvilli within these dendritic processes, indicating ductal differentiation. Immunocytochemically, the dendritic processes were positive for cytokeratin 19, a marker for bile duct cells. On the other hand, an H-ras-transformed mouse liver epithelial cell line and rat hepatocellular carcinoma cell lines did not demonstrate the organized morphogenesis. Our results indicate that hepatocytic cells can produce bile duct-like structures in the presence of the type I collagenous matrix and soluble morphogenetic factors.     

Fibroblasts Stimulate Epithelization of Collagen Gel

We studied the influence of human embryonic fibroblasts on epithelization of collagen gel. Introduction of the fibroblasts into the gel stimulates proliferation of keratinocytes plated on the gel surface. The presence of fibroblasts in the gel also affects the pattern of keratinocyte migration on the gel and induces pronounced polarization of the migrating colonies.     

Epithelial Sheet Folding Induces Lumen Formation by Madin-Darby Canine Kidney Cells in a Collagen Gel

Lumen formation is important for morphogenesis; however, an unanswered question is whether it involves the collective migration of epithelial cells. Here, using a collagen gel overlay culture method, we show that Madin-Darby canine kidney cells migrated collectively and formed a luminal structure in a collagen gel. Immediately after the collagen gel overlay, an epithelial sheet folded from the periphery, migrated inwardly, and formed a luminal structure. The inhibition of integrin-β1 or Rac1 activity decreased the migration rate of the peripheral cells after the sheets folded. Moreover, lumen formation was perturbed by disruption of apical-basolateral polarity induced by transforming growth factor-β1. These results indicate that cell migration and cell polarity play an important role in folding. To further explore epithelial sheet folding, we developed a computer-simulated mechanical model based on the rigidity of the extracellular matrix. It indicated a soft substrate is required for the folding movement.     

Epithelial-Mesenchymal Transition Stimulates Human Cancer Cells to Extend Microtubule-based Invasive Protrusions and Suppresses Cell Growth in Collagen Gel

Epithelial-mesenchymal transition (EMT) is a crucial event in tumor invasion and metastasis. However, most of past EMT studies have been conducted in the conventional two-dimensional (2D) monolayer culture. Therefore, it remains unclear what invasive phenotypes are acquired by EMT-induced cancer cells. To address this point, we attempted to characterize EMT cells in more physiological, three-dimensional (3D) collagen gel culture. EMT was induced by treating three human carcinoma cell lines (A549, Panc-1 and MKN-1) with TGF-ß. The TGF-ß treatment stimulated these cells to overexpress the invasion markers laminin γ2 and MT1-MMP in 2D culture, in addition to the induction of well-known morphological change and EMT marker expression. EMT induction enhanced cell motility and adhesiveness to fibronectin and collagen in 2D culture. Although EMT cells showed comparable cell growth to control cells in 2D culture, their growth rates were extremely suppressed in soft agar and collagen gel cultures. Most characteristically, EMT-induced cancer cells commonly and markedly extended invasive protrusions in collagen gel. These protrusions were mainly supported by microtubules rather than actin cytoskeleton. Snail-introduced, stable EMT cells showed similar protrusions in 3D conditions without TGF-ß. Moreover, these protrusions were suppressed by colchicine or inhibitors of heat shock protein 90 (HSP-90) and protein phosphatase 2A. However, MMP inhibitors did not suppress the protrusion formation. These data suggest that EMT enhances tumor cell infiltration into interstitial stroma by extending microtubule-based protrusions and suppressing cell growth. The elevated cell adhesion to fibronectin and collagen and high cell motility also seem important for the tumor invasion.     

Simulation of Migration of Human Epidermal Keratinocytes over Three-Dimensional Collagen Gel

We developed a novel model for studying migration of keratinocyte colonies over 3D collagen gel. It was shown that keratinocytes previously cultured on plastic and then seeded on the surface of collagen gel proliferate more actively than the primary cells. The gel density could significantly affect the morphology and migration of keratinocyte colonies. Modification of collagen gel by fibronectin-like protein or poly-L-lysine altered the morphometric parameters of colonies without significant changes in the velocity of migration.     

Modulation of Heart Fibroblast Migration and Collagen Gel Contraction by IGF-I

Dynamic interactions between cells and the extracellular matrix are essential in the regulation of a number of cellular processes including migration, adhesion, proliferation and differentiation. A variety of factors have been identified which modulate these interactions including transforming growth factor+, platelet-derived growth factor and others. Insulin-like growth factors have been shown to regulate collagen production by heart fibroblasts; however, the effects of this growth factor on the interactions of heart fibroblasts with the extracellular matrix have not been examined. The present studies were carried out to determine the effects of IGF-I on the ability of fibroblasts to interact with the extracellular matrix and to begin to determine the mechanisms of this response. These experiments illustrate that IGF-I treatment results in increased migration, collagen reorganization and gel contraction by heart fibroblasts. IGF-I has been shown to activate both the mitogen-activated protein kinase and phophatidylinositol-3 kinase pathways in isolated cells. Experiments with pharmacological antagonists of these pathways indicate that the mitogen-activated protein kinase pathway is essential for IGF-I stimulated collagen gel contraction by fibroblasts. These studies illustrate that IGF-I modulates the ability of fibroblasts to interact with the collagen matrix and that activation of multiple signaling pathways by IGF-I may produce distinct downstream responses in these cells.     

Scalable Fabrication of Efficient Perovskite Solar Modules on Flexible Glass Substrates

Perovskite materials are good candidates for flexible photovoltaic applications due to their strong absorption and low‐temperature processing, but efficient flexible perovskite modules have not yet been realized. Here, a record efficiency flexible perovskite solar module is demonstrated by blade coating high‐quality perovskite films on flexible Corning Willow Glass using additive engineering. Ammonium chloride (NH₄Cl) is added into the perovskite precursor solution to retard the nucleation which prevents voids formation at the interface of perovskite and glass. The addition of NH₄Cl also suppresses the formation of PbI₂ and reduces the trap density in the perovskite films. The implementation of NH₄Cl enables the fabrication of single junction flexible perovskite solar devices with an efficiency of 19.72% on small‐area cells and a record aperture efficiency of 15.86% on modules with an area of 42.9 cm². This work provides a simple way to scale up high‐efficiency flexible perovskite modules for various applications.     

Effect of Collagen Gel Configuration on the Cytoskeleton in Cultured Rat Hepatocytes

The cytoskeleton is important in the maintenance of cellular morphology and differentiated function in a number of cell types, including hepatocytes. In this study, adult rat hepatocytes sandwiched between two layers of collagen gel were compared to cells cultured on a single collagen gel for differences in the organization and expression of the cytoskeletal proteins actin and tubulin. Hepatocytes cultured between two layers of hydrated rat tail tendon collagen (sandwich gel) morphologically resembled cells in intact liver for several weeks. Actin filaments (F-actin) in these hepatocytes were concentrated under the plasma membrane in regions of cell-cell contact. In contrast, hepatocytes cultured on a single collagen gel were flattened and motile and had F-actin containing stress fibers. This was accompanied by a severalfold increase in actin mRNA. Microtubules formed an interwoven network in hepatocytes cultured in a sandwich gel, but in single gel cultures they formed long parallel arrays extending out to the cell periphery. Tubulin mRNA was severalfold greater in hepatocytes cultured on a single gel. Fibronectin and laminin staining were greater in single gel cultures, and these proteins were concentrated in fibrils radiating from the cell periphery. Overlaying a second collagen gel onto hepatocytes that had been cultured on a single gel (double gel rescue) reversed cell spreading and reduced stress fibers. Double gel rescue also resulted in a decrease in actin and tubulin mRNA to levels present in sandwich gel cultures and freshly isolated hepatocytes. These results show that the configuration of the external matrix has a dynamic effect on cytoskeletal proteins in cultured rat hepatocytes.     

Accelerating Effect of Soy Peptides Containing Collagen Peptides on Type I and III Collagen Levels in Rat Skin

Two weeks of feeding soy peptides containing 2% collagen peptides increased the levels of type I and III tropocollagen and their mRNAs. In contrast, the diet did not increase the mRNA levels of rat hyaluronan synthases, serine palmitoyltransferase (the rate-limiting enzyme of ceramide synthesis), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (the key enzyme of cholesterol synthesis). These results suggest that feeding of soy peptides with collagen peptides specifically enhanced the tropocollagen level in the skin.     

Growth and Differentiation of Primary Cultures of Mouse Mammary Epithelium Embedded in Collagen Gel

Mammary glands from BALB/cfC3H midpregnant (9–11 days) mice were dissociated with collagenase and pronase, separated on a Percoll gradient, and the epithelial cells were cultured inside collagen gel. The cell number increased three-to five-fold when cultured for 6–8 days in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), cortisol (1.0 μg/ml), prolactin (10 μg/ml), transferrin (10 μg/ml), and epidermal growth factor (0.01 μg/ml). The casein level, as determined by radioimmunoassay, at the end of this growth phase, was much lower than that present in freshly dissociated cells. In order to stimulate casein production, the gels were released from the sides of the plastic dish and allowed to float for eight days in Waymouth's medium, containing insulin (10 μg/ml), cortisol (5 μg/ml), prolactin (10 μg/ml), and 0.25% bovine serum albumin. The casein level at the end of this differentiation phase was found to be comparable to that seen in the original freshly dissociated cells. Cells grown in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), and transferrin (10 μg/ml) were still capable of undergoing casein production, indicating that the presence of exogenous lactogenic hormones such as cortisol and prolactin, as well as exogenous growth factors such as epidermal growth factor, is not necessary during the growth phase for subsequent casein production during the differentiation phase. Two factors that seemed more important for subsequent casein stimulation were: (1) releasing collagen gels at the beginning of the differentiation phase, and (2) switching to'differentiation' medium. This present two-step protocol has allowed primary cultures of dissociated midpregnant mouse mammary epithelial cells to undergo several rounds of division inside a collagen gel matrix and to be subsequently stimulated to produce the mammary-specific protein, casein.     

Collagen targeting using multivalent protein-functionalized dendrimers

Collagen is an attractive marker for tissue remodeling in a variety of common disease processes. Here we report the preparation of protein dendrimers as multivalent collagen targeting ligands by native chemical ligation of the collagen binding protein CNA35 to cysteine-functionalized dendritic divalent (AB₂) and tetravalent (AB₄) wedges. The binding of these multivalent protein constructs was studied on collagen-immobilized chip surfaces as well as to native collagen in rat intestinal tissues. To understand the importance of target density we also created collagen-mimicking surfaces by immobilizing synthetic collagen triple helical peptides at various densities on a chip surface. Multivalent display of a weak-binding variant (CNA35-Y175K) resulted in a large increase in collagen affinity, effectively restoring the collagen imaging capacities for the AB₄ system. In addition, dissociation of these multivalent CNA35 dendrimers from collagen surfaces was found to be strongly attenuated.     

Simultaneous Immunofluorescent Detection of Coentrapped Cells in Gel Beads

An immunofluorescent method involving double color labeling and confocal microscopy was reported to specifically detect lactic acid bacteria and probiotic cells coimmobilized in gels beads. The method described is rapid (4 h) and sensitive and may be useful for studying cell dynamics during mixed-culture starter production using immobilized cells in gel beads. Microscopic observations were perfectly correlated to cell counts obtained using a sandwich enzyme-linked immunosorbent assay.     

Microstructural Modeling of Collagen Network Mechanics and Interactions with the Proteoglycan Gel in Articular Cartilage

Cartilage matrix mechanical function is largely determined by interactions between the collagen fibrillar network and the proteoglycan gel. Although the molecular physics of these matrix constituents have been characterized and modern imaging methods are capable of localized measurement of molecular densities and orientation distributions, theoretical tools for using this information for prediction of cartilage mechanical behavior are lacking. We introduce a means to model collagen network contributions to cartilage mechanics based upon accessible microstructural information (fibril density and orientation distributions) and which self-consistently follows changes in microstructural geometry with matrix deformations. The interplay between the molecular physics of the collagen network and the proteoglycan gel is scaled up to determine matrix material properties, with features such as collagen fibril pre-stress in free-swelling cartilage emerging naturally and without introduction of ad hoc parameters. Methods are developed for theoretical treatment of the collagen network as a continuum-like distribution of fibrils, such that mechanical analysis of the network may be simplified by consideration of the spherical harmonic components of functions of the fibril orientation, strain, and stress distributions. Expressions for the collagen network contributions to matrix stress and stiffness tensors are derived, illustrating that only spherical harmonic components of orders 0 and 2 contribute to the stress, while orders 0, 2, and 4 contribute to the stiffness. Depth- and compression-dependent equilibrium mechanical properties of cartilage matrix are modeled, and advantages of the approach are illustrated by exploration of orientation and strain distributions of collagen fibrils in compressed cartilage. Results highlight collagen-proteoglycan interactions, especially for very small physiological strains where experimental data are relatively sparse. These methods for determining matrix mechanical properties from measurable quantities at the microscale (composition, structure, and molecular physics) may be useful for investigating cartilage structure-function relationships relevant to load-bearing, injury, and repair.     

Phenotypic Modulation of Hamster Acinar Cells by Culture in Collagen Matrix

The aim of this study was to assess the effect of different culture conditions on the survival and morphological phenotype of cultured acinar cells. Acinar fragments isolated from hamster pancreas were embedded in rat-tail collagen. Four groups were established: Medium 1—5% NuSerum + basic medium (basic MEDIUM = DMEM/F12 supplemented with dexamethasone, 3-isobutyl-2-methylxanthine, and antibiotics); Medium 2—10% NuSerum + basic medium. Medium 3—Medium 2 supplemented with epidermal growth factor and cholera toxin; and Medium 4:—Medium 3 supplemented with soybean trypsin inhibitor. Freshly isolated acinar cells were retrieved morphologically intact. In Medium 1, more than 80% of cells retained a normal histological appearance at 34 days in culture. Immunostaining for amylase was observed at the apical pole of the cells. The remaining cells showed variable degrees of degeneration. In Medium 2, approximately 50% of acinar cells appeared normal at 34 days in culture, while the remainder were severely degenerated. A few cystic structures were also observed. Positive immunostaining for amylase was limited to the cells with a normal histological appearance. The cells grown in Media 3 and 4 had similar courses of morphological changes. After 8 days in culture, most acinar fragments disappeared and were replaced by cystic structures, lined by a single layer of cuboidal cells. Some amylase-positive immunoreactive cells were integral components of the cystic wall. Cellular amylase activity was a function of the different culture media, a more rapid decrease in amylase activity being observed in Media 3 and 4. Uptake of [³H]thymidine did not show any significant differences between the media. It was also found that the ductlike cells cultured in Medium 4 had a limited capacity to redifferentiate into acinar cells. This study shows that the acinar cell phenotype can be maintainedin vitrofor more than 1 month. This study also suggests that ductal-like epithelial structures arise from transformation of acinar cells.     

Fluoride Ingestion After Brushing with a Gel Containing a High Concentration of Fluoride

The aim of the study was to evaluate the amount of fluoride remaining in the oral cavity of children after brushing with fluoride gel (1.25% F). The study involved six groups of 7-year-old and six groups of 11-year-old children. The procedure was carried out according to the manufacturer’s recommendations. Fluoride concentrations were determined using ion-selective fluoride electrode. No statistically significant difference was found between the amount of fluorides that remained in the oral cavity of younger and older age group (1.2 and 1.3 mg, respectively; p > 0.05). The amount of fluorides swallowed during the procedure in both age groups proves to be within acceptable limit, as far as risk of acute poisoning symptoms is concerned. The individual daily fluoride exposure during the day of procedure seems to be twice as high compared to average fluoride intake from diet and dentifrice, and it does not exceed Tolerable Upper Intake Level for children more than 8. In younger children, it seems justifiable to reduce the amount of the preparation applied on a toothbrush, especially when daily use of the gel is recommended.     

Involvement of Collagen Synthesis in Tissue Reconstitution by Dissociated Sponge Cells

Cells dissociated from the sponge Haliclona permollis reconstituted a new body by the following three consecutive processes: (1) Reaggregation, cells aggregate as a spheroidal mass. (2) Spreading, aggregates flatten and spread. (3) Reconstruction, aggregates coalesce into a reconstituted body. Cells in the process of reconstitution incorporated ¹⁴C-proline into proteins, converting 17% of it to hydroxyproline and synthesize collagen molecules during this process. Inhibitors of collagen biosynthesis did not affect the reaggregation, but caused incomplete morphogenesis in the processes of spreading and reconstruction: cycloheximide and 2,2'-dipyridyl inhibited spreading while 3-aminopropionitrile induced incomplete reconstruction. These findings suggested that spreading and reconstruction, but not reaggregation require both synthesis and cross-linking of collagens. Three polypeptides with molecular weights of 58 K, 160 K and 180 K were identified in sponge cells as collagens by immunoblot analysis with antibodies against sea urchin collagen and studies on susceptibilities to collagenase and pepsin. The 58 KDa polypeptide appeared in reconstituted bodies but not in dissociated cells, suggesting its importance in tissue reconstitution by dissociated cells.     

Electrospun Scaffold of Collagen and Polycaprolactone Containing ZnO Quantum Dots for Skin Wound Regeneration

Nanofibers(NFs)have been widely used in tissue engineering such as wound healing.In this work,the antibacterial ZnO quantum dots(ZnO QDs)have been incorporated into the biocompatible poly(ε-caprolactone)/collagen(PCL/Col)fibrous scaffolds for wound healing.The as-fabricated PCL-Col/ZnO fibrous scaffolds exhibited good swelling,antibacterial activity,and biodegradation behaviors,which were beneficial for the applications as a wound dressing.Moreover,the PCL-Col/ZnO fibrous scaffolds showed excellent cytocompatibility for promoting cell proliferation.The resultant PCL-Col/ZnO fibrous scaffolds containing vascular endothelial growth factor(VEGF)also exhibited promoted wound-healing effect through promoting expression of transforming growth factor-β(TGF-β)and the vascular factor(CD31)in tissues in the early stages of wound healing.This new electrospun fibrous scaffolds with wound-healing promotion and antibacterial property should be convenient for treating wound healing.     

Study of Intracellular Collagen Precursors using DNA-Cellulose Chromatography

PREVIOUS communications^1,2 have described the synthesis in cultured cells of proteins with affinity for columns of denatured DNA-cellulose³. Polyacrylamide gel electrophoresis of the proteins eluted from such columns by 0.15 M NaCl has shown differences between the DNA-cellulose binding proteins synthesized by growing and resting fibroblasts^1,2. One protein, P-6, later shown to have a molecular weight in SDS of 40,000, was synthesized only by growing cells, while P-1 and P-1′, much larger proteins, were very prominent only in cells resting due to serum limitation and contact inhibition. Investigation of the nature and role of P-6 and other proteins synthesized by growing cells is continuing; as for the DNA binding proteins synthesized by resting cells, we report here that P-1 and P-1′ do not seem to be related to growth control, as suggested by the earlier experiments, but are collagen type molecules, probably larger molecular weight protocollagen precursors of the α₁ and α₂-chains respectively.     

Use of Collagen Gel as a Three-Dimensional In Vitro Model to Study Electropermeabilization and Gene Electrotransfer

Gene electrotransfer is a promising nonviral method that enables transfer of plasmid DNA into cells with electric pulses. Although many in vitro and in vivo studies have been performed, the question of the implied gene electrotransfer mechanisms is largely open. The main obstacle toward efficient gene electrotransfer in vivo is relatively poor mobility of DNA in tissues. Since cells are mechanically coupled to their extracellular environment and act differently compared to standard in vitro conditions, we developed a three-dimensional (3-D) in vitro model of CHO cells embedded in collagen gel as an ex vivo model of tissue to study electropermeabilization and different parameters of gene electrotransfer. For this purpose, we first used propidium iodide to detect electropermeabilization of CHO cells embedded in collagen gel. Then, we analyzed the influence of different concentrations of plasmid DNA and pulse duration on gene electrotransfer efficiency. Our results revealed that even if cells in collagen gel can be efficiently electropermeabilized, gene expression is significantly lower. Gene electrotransfer efficiency in our 3-D in vitro model had similar dependence on concentration of plasmid DNA and pulse duration comparable to in vivo studies, where longer (millisecond) pulses were shown to be more optimal compared to shorter (microsecond) pulses. The presented results demonstrate that our 3-D in vitro model resembles the in vivo situation more closely than conventional 2-D cell cultures and, thus, provides an environment closer to in vivo conditions to study mechanisms of gene electrotransfer.