Synthesis and decoloring properties of sodium humate/poly (N-isopropylacrylamide) hydrogels

A series of novel sodium humate/poly(N-isopropylacrylamide) (SH/PNIPA) hydrogels were synthesized by solution polymerization. The swelling and decoloring properties of SH/PNIPA hydrogels were also examined. Experiment results show that there exist hydrogen-bonding interactions between SH and PNIPA in the SH/PNIPA hydrogels network, which are not strong enough to disrupt the aggregation of dehydrated PNIPA chains at phase transition temperature, leading to the same volume phase transition temperature as pure PNIPA hydrogel. The adsorption and desorption of methylene blue (MB) for the hydrogels were influenced by temperature, initial MB concentration and SH amount. Low temperature favors the adsorption and desorption of MB. Appropriate SH amount of the hydrogels is crucial for the adsorption and desorption of MB. The maximum adsorption capacity was 10.8 mg MB per gram of SH/PNIPA gel.     

Swelling and de-swelling kinetics of gelatin hydrogels in ethanol-water marginal solvent

Controlled osmotic swelling and de-swelling measurements have been performed on gelatin, a polyampholyte, hydrogels suspended in water-ethanol marginal solvent at room temperature (20 degrees C) where the alcohol concentration was changed from 0 to 100% (v/v). The change in gel mass was monitored as function of time until osmotic equilibrium was established with the surrounding solvent. It was observed that osmotic pressure of polymer-solvent mixing, pi(m)相似文献   

Mechanisms controlling the temperature-dependent binding of proteins to poly(N-isopropylacrylamide) microgels

Poly(N-isopropylacrylamide) (PNIPA) microgels may offer several advantages over PNIPA-modified surfaces when used as sorbents in temperature-sensitive chromatography. Yet, a full exploitation of these advantages requires a better understanding of the mechanisms controlling the separation process. As a model system, we have studied the binding of three proteins (bovine serum albumin (BSA), ovalbumin, and lysozyme) to PNIPA microgels. Binding experiments were conducted both below (25 degrees C) and above (37 degrees C) the volume phase transition temperature of the gel, T(c). The analysis of the binding isotherms has shown that although an average gel particle contained a larger amount of protein below the phase transition temperature, the concentration of the protein within the particle was higher above this temperature. These findings were attributed to changes in the binding loci due to temperature swings around T(c): whereas a sorption mechanism is dominant below this temperature, surface-adsorption was more important above it. A comparison between the three studied proteins has shown that below T(c) the binding increases with a decrease in the molecular weight. On the other hand, no significant difference in the bound protein amounts was observed above the phase transition temperature. Our results imply that, despite the increase in the gel's hydrophobicity above the phase transition temperature, the resolution in bioseparations based on PNIPA gels is not necessarily better above T(c).     

Preparation and characterization of imogolite/DNA hybrid hydrogels

Imogolite is one of the clay minerals contained in volcanic ash soils. The novel hybrid hydrogels were prepared from imogolite nanofibers and DNA by utilizing strong interaction between the aluminol groups on imogolite surface and phosphate groups of DNA. The hybrid hydrogels of imogolite and DNA were prepared in various feed ratios, and their physicochemical properties and molecular aggregation states were investigated in both dispersion and gel states. The maximum DNA content in the hybrid gels was shown in equivalent molar ratio of imogolite and DNA. The physical properties of the hybrid gels were changed by varying DNA blend ratios. In the dispersion state, the hybrid gels showed a fibrous structure of imogolite, whereas a continuous network structure was observed in pure imogolite, indicating that the hybrid with DNA enhanced the dispersion of imogolite. In the gel state, DNA and imogolite nanofibers formed a 3D network structure.     

Cell proliferation and cell sheet detachment from the positively and negatively charged nanocomposite hydrogels

The charged nanocomposite hydrogels (NC gels) were synthesized by copolymerization of positively or negatively chargeable monomer with N‐isopropylacrylamide (NIPAm) in the aqueous suspension of hectorite clay. The ionic NC gels preserved the thermo‐responsibility with the phase‐transition temperature below 37°C. The L929 cell proliferation was sensitive to charge polarity and charge density. As compared to the PNIPAm NC gel, the cationic NC gels with <5 mol % of 2‐(dimethylamino)ethyl methacrylate (DMAEMA) showed improved cell proliferation, whereas the cells grew slowly on the gels with negatively charged 2‐acrylamido‐2‐methylpropane sulfonic acid (AMPSNa). By lowering temperature, rapid cell sheet detachment was observed from the surface of ionic NC gels with 1 mol % of ionizable monomers. However, lager amount of AMPSNa or DMAEMA did not support rapid cell sheet detachment, probably owing to the adverse swelling effects and/or enhanced electrostatic attraction. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 58–65, 2014.     

Cell adhesion and proliferation on poly(N-vinylacetamide) hydrogels and double network approaches for changing cellular affinities

Poly(N-vinylacetamide) hydrogels (PNVA gels) were synthesized to investigate their basic characteristics for biomedical applications such as water contact angles, protein uptake, and mouse fibroblasts (L-929) cell adhesion. Because PNVA gels show hydrophilic features, double network (DN) hydrogels were prepared by the secondary polymerization of N-vinylacetamide (NVA) or acrylamide (AAm) in PNVA gels (NVA/NVA DN gels and NVA/AAm DN gels, respectively), in order to vary PNVA gel features for biocompatibility. Contact angles for both DN gels decreased to around 20 degrees, whereas both PNVA and PAAm gels were over 30 degrees. On the other hand, more protein tended to adsorb to DN gels than single network hydrogels. Compared to PNVA gel, cell adhesion and proliferation on NVA/NVA DN gel were improved with less swelling ratio and much protein uptake, while no significant difference was observed on NVA/AAm DN gel, probably due to more hydrophilic character, supported by lowest water contact angle. These complicated structure change in DN gels would provide a new methodology for tuning the biocompatibility of hydrogels and for controlling surface hydrophilic characteristics and network structures.     

Biodegradable and biocompatible synthetic saccharide-Peptide hydrogels for three-dimensional stem cell culture

Saccharide-peptide hydrogels have been developed in our laboratory as new synthetic extracellular matrices for regenerative medicine applications. In this work, we have expanded on our previously reported system and applied copolymerization of cysteine (Cys) and vinyl sulfone (VS)-functionalized saccharide-peptide polymers via Michael-type addition for encapsulation and 3D culture of cells. Specifically, our aims were to (1) develop a novel hydrogel platform, which could be applied for encapsulating and culturing mesenchymal stem cells (MSCs) in a 3D environment, (2) characterize the tunable properties of the hydrogel, specifically, degradation, mechanical, and gel network properties, and (3) determine the biocompatibility of the saccharide-peptide hydrogel material with MSCs. Hydrogel mechanical properties were tunable by varying the VS:Cys ratio (= 0.5, 1, or 2) as well as the pH (6, 7, or 8) of the cross-linking components. Stiffer gels were formed at VS:Cys = 1 and pH 6 or 7. Gels formed at pH 8 or with excess Cys (VS:Cys = 0.5) or VS (VS:Cys = 2) were significantly softer. Cross-linking pH and VS:Cys ratio also had an effect on the degradation behavior of the VS:Cys gels, with higher cross-linking pH resulting in an accelerated loss of mass. On the basis of environmental scanning electron microscopy (ESEM) analysis and fluorescence microscopy, all hydrogels appeared to exhibit porous gel networks. MSCs cultured in monolayer and exposed to soluble Cys or VS copolymers (0.1-5 mg/mL) did not exhibit measurable cytotoxicity. In addition, MSCs were cultured in 3D for up to 14 days in vitro without deleterious effects on cell viability. In summary, we have established and characterized a tunable 3D saccharide-peptide hybrid copolymer hydrogel platform for culturing MSCs. Future studies will focus on utilizing the hydrogel system for controlling the differentiation of MSCs.     

The use of colloidal gold complexes in an ultrastructural study of lectin-binding sites and matrix deposition of normal human primary breast epithelium on collagen gels

Summary Colloidal gold probes were used to study the distribution of peanut agglutinin binding sites and the deposition of extracellular fibronectin and type IV collagen in cultured human breast cells grown on type I collagen gels. Qualitative analysis was performed at the ultrastructural level and appraised in relation to the possible role of peanut agglutinin, fibronectin and type IV collagen as functional markers for distinguishing cell types using this methodology.Peanut agglutinin bound to the surface of cuboidal epithelial cells but not on basal, putative myoepithelial cells in the cell islands, suggesting that it may be a useful functional marker. The binding on the epithelial cells was markedly increased by pre-treatment of the cells with neuraminidase. No correlation was seen between the amount of binding and either the surface topography or cellular ultrastructure.Fibronectin and type IV collagen were demonstrated on the fibrillar network left on the collagen gels after removal of the cell sheet. Any cells still adhering to the gel surface showed no evidence of gold probe binding on their upper surfaces. Examination of the under surfaces of the cell sheet showed gold probe binding equivalent to that found on the gels under the cells. However, it was not proven conclusively which cells produce the fibronectin and type IV collagen.     

Influence of the extracellular matrix on the proliferative response of human skin fibroblasts to serum and purified platelet-derived growth factor

The culture of adult human skin fibroblasts on reconstituted bovine type 1 fibrillar collagen gels, ranging in concentration from 2.5-35.0 mg/ml, results in a reduction in proliferation rate by 40%-60% as measured by (3H) thymidine incorporation. The suppressive effect was noted when cells were cultured in both human and bovine serum. Drying the gels into thin films abolishes the suppressive effect of the fibrillar collagen on cell proliferation. Cell attachment studies showed that differences in the proliferation rate of cells on the various substrata were not simply due to differences in initial attachment. Studies with purified platelet-derived growth factor (PDGF) demonstrated that the reduced responsiveness of cells to this factor, when cultured on collagen gels as compared to plastic, was largely responsible for the reduced proliferative activity of the cells when cultured in the presence of serum. The reduced proliferative activity of fibroblasts in response to PDGF, when cultured on collagen gels, was confirmed by total DNA determination. It was shown that the reduced responsiveness of cells to PDGF was not simply because the factor bound to the fibrillar collagen gel or was inaccessible to the cells. The data indicate that the reduced proliferation rate of fibroblasts cultured on collagen gels is a direct result of the influence of the extracellular matrix on the cells' ability to respond to a soluble mitogenic mediator.     

Effect of protein-hydroxyethylmethacrylate hydrogels on cultured endothelial cells

The use of protein hydroxy ethylmethacrylate (HEMA) hydrogels to control cell morphology and growth, as well as the synthesis of extracellular matrix components, is described in this communication. HEMA hydrogels prepared with collagen support growth of embryonic lung fibroblasts (IMR-90), as well as bovine aortic and pulmonary artery endothelial cells at a level comparable to the respective cells grown on tissue culture surfaces. On the other hand, HEMA hydrogels prepared with solubilized elastin inhibit the fibroblast growth and prevent both types of endothelial cell cultures from achieving their normal morphology. These morphologically altered endothelial cells resume a normal cobblestone-like appearance when subcultivated from the elastin-HEMA hydrogels to tissue culture plastic. When pulsed with [14C]proline, the procollagens synthesized by the endothelial cells on the different surfaces vary, as shown by immunoprecipitation and polyacrylamide gel electrophoresis. On the standard tissue culture plastic, the confluent cells produce mainly type III procollagen in the medium, whereas those endothelial cells grown on collagen and elastin-HEMA hydrogels synthesize primarily type I procollagen (much like sprouting cells on tissue culture plastic), regardless of their morphology.     

CELL POSITION-DEPENDENT RECIPROCAL FEEDBACK REGULATION OF TYPE I COLLAGEN GENE EXPRESSION IN CULTURED HUMAN SKIN FIBROBLASTS

In order to investigate possible cell positional effects on the gene expression of human dermal fibroblasts, the authors cultured the cells on non-coated polystyrene culture dishes, type I collagen-coated dishes, or collagen gels formed by type I collagen, or suspended them in type I collagen gels and measured collagen synthesis by the cells. The production rate of type I collagen was similar whether cells were cultured on non-coated polystyrene or on type I collagen-coated dishes, but it was suppressed significantly when the cells were placed within the collagen gel matrix. Time-dependent expression of genes for α1(I) and α2(I) collagen chains was measured by Northern blot analysis. A significant increase in mRNA levels for these chains was observed when the cells were cultured for three days on type I collagen-coated dishes or on collagen gels. On the other hand, a significant decrease in the mRNA levels was observed after 2 days and later, when the cells were cultured within type I collagen gel matrix. These results indicate that human dermal fibroblasts recognize their position on or in type I collagen (extracellular matrix) and respond by changing their expression patterns of type I collagen chain genes. The results of the kinetics of gene expression also suggest that upregulation and downregulation of type I collagen genes are controlled by different mechanisms.     

Microporous cell‐laden hydrogels for engineered tissue constructs

In this article, we describe an approach to generate microporous cell‐laden hydrogels for fabricating biomimetic tissue engineered constructs. Micropores at different length scales were fabricated in cell‐laden hydrogels by micromolding fluidic channels and leaching sucrose crystals. Microengineered channels were created within cell‐laden hydrogel precursors containing agarose solution mixed with sucrose crystals. The rapid cooling of the agarose solution was used to gel the solution and form micropores in place of the sucrose crystals. The sucrose leaching process generated homogeneously distributed micropores within the gels, while enabling the direct immobilization of cells within the gels. We also characterized the physical, mechanical, and biological properties (i.e., microporosity, diffusivity, and cell viability) of cell‐laden agarose gels as a function of engineered porosity. The microporosity was controlled from 0% to 40% and the diffusivity of molecules in the porous agarose gels increased as compared to controls. Furthermore, the viability of human hepatic carcinoma cells that were cultured in microporous agarose gels corresponded to the diffusion profile generated away from the microchannels. Based on their enhanced diffusive properties, microporous cell‐laden hydrogels containing a microengineered fluidic channel can be a useful tool for generating tissue structures for regenerative medicine and drug discovery applications. Biotechnol. Bioeng. 2010; 106: 138–148. © 2010 Wiley Periodicals, Inc.     

Tumor cell haptotaxis on covalently immobilized linear and exponential gradients of a cell adhesion peptide

The movement of cells up an adhesive substratum gradient has been proposed as a mechanism for directing cell migration during development and metastasis. Critical evaluation of this hypothesis (haptotaxis) benefits from the use of quantifiable, stable substratum gradients of biologically relevant adhesion molecules. We report covalent derivatization of polyacrylamide surfaces with quantifiable gradients of a nonapeptide containing the adhesive Arg-Gly-Asp sequence. Cell migration was studied by seeding derivatized surfaces evenly with B16F10 murine melanoma cells. Within 8 hr, cells on gradients redistributed markedly; higher cell densities were found at gel positions having higher immobilized peptide densities. In contrast, cells seeded on control gels with uniform concentrations of adhesive peptide did not redistribute. Redistribution occurred on gradients in both serum-free and serum-containing media. Experiments with uniform density peptide-derivatized gels demonstrated that redistribution on gradients was not due to preferential initial cell attachment or preferential growth on the higher density of immobilized peptide, but must have been due to cell translocation. Cells on exponential gradients of immobilized peptide migrated to a position on the gel surface corresponding to the highest immobilized peptide density, while cells on linear gradients of the same peptide migrated to a position of intermediate peptide density. These data suggest that the B16F10 cells respond to proportional changes in immobilized peptide density rather than to absolute changes, implying a sensing mechanism which utilizes adaptation. These results demonstrate that (1) a gradient of a small adhesive peptide is sufficient to generate redistribution of cell populations and (2) controlled quantifiable substratum gradients can be produced and used to probe the underlying cellular mechanisms of this behavior.     

The influence of cell shape on the induction of functional differentiation in mouse mammary cells in vitro

Summary To define more clearly the in vitro conditions permissive for hormonal induction of functional differentiation, we cultured dissociated normal mammary cells from prelactating mice in or on a variety of substrates. Cultivation of an enriched epithelial cell population in association with living adult mammary stroma in the presence of lactogenic hormones resulted in both morphological and biochemical differentiation. This differentiation, however, was not enhanced over that seen when the cells were associated with killed stroma, provided that the killed stroma had a flexibility similar to that of the living stroma. Cells cultured in inflexible killed stroma usually did not differentiate. Cells cultured within the flexible environment of a collagen gel, but removed from the gas-medium interface, differentiated in a manner similar to those cultured in flexible stroma. Cells cultured on the surface of an attached collagen gel were squamous, and their basolateral surfaces were sequestered from the medium; they did not differentiate. Cells cultured on floating collagen gels were cuboidal-columnar, with basolateral surfaces exposed to the medium, and showed good functional differentiation. Cells cultured on inflexible floating collagen gels were extremely flattened and had exposed basolateral surfaces, and showed no evidence of functional differentiation. We infer that assumption of cuboidal to columnar shapes similar to those of mammary cells in vivo may be important to the induction of functional differentiation in vitro. The additional requirement of basolateral cell surface exposure also is important. This work was supported by U.S. Public Health Service Grants CA-05045 and CA-09041 from the National Cancer Institute, Bethesda, MD.     

Molecular dynamics study of the influence of solvents on the structure and mechanical properties of poly(vinyl alcohol) gels

Molecular dynamics (MD) simulations were employed to study the influence of solvents on the structure and mechanical properties of physically crosslinked poly(vinyl alcohol) (PVA) gels. Firstly, three kinds of PVA precursor gels were made by adding water, dimethyl sulfoxide (DMSO) and a mixture of DMSO and water (4:1 by weight), respectively. The solvents in the precursor gels were then exchanged with water to obtain three kinds of PVA hydrogels. Solvent in the precursor gel with a mixture of DMSO and water was also exchanged with ethanol and DMSO, respectively. It was found that the tensile strength and failure strain of the PVA hydrogel prepared from precursor gel with a mixture of DMSO and water was the highest, and the polymer network was more homogeneous than the other two PVA hydrogels. The polymer network of PVA gel with ethanol or with DMSO was more heterogenous than with water, and the tensile strength and failure strain were much lower. The torsional activity of polymer chains of PVA gel with ethanol was much stronger than PVA gel with water and DMSO.     

Maintenance of stem cell viability and differentiation potential following cryopreservation within 3-dimensional hyaluronic acid hydrogels

While significant progress has been made in directing the behavior of cells encapsulated within three-dimensional (3D) covalently crosslinked hydrogels, the capacity of these materials to support in situ cryopreservation of cells directly within the gels has not been assessed. Here, we demonstrate the retention of human mesenchymal stem cell (hMSC) viability within hyaluronic acid (HA) and polyethylene glycol based hydrogels via a facile gradual cooling and freezing protocol. Encapsulated cell viability was retained at similar rates in both materials systems regardless of initial duration in culture or adhesive ligand incorporation, indicating the versatility of the approach. Additionally, the cryopreservation protocol maintains stem cell differentiation potential; incubation in adipogenic differentiation media induced equal rates of hMSC adipogenesis in freeze-thawed and non-frozen HA based hydrogels on a per-cell basis. Collectively, these findings highlight the cryopreservation protocol as a platform technology that, in addition to contributing to an increased understanding of three-dimensional cell-matrix interactions, could enable the long-term preservation of tissue engineering constructs for clinical applications.     

Temperature-dependent cell detachment on Pluronic gels

Cell culture on gels made of poly(ethylene oxide) and poly(propylene oxide) (Pluronic), which has a lower critical solution temperature around 30 degrees C, could be performed for 48 h. However, the Pluronic gels were highly hydrophilic and tended to dissolve in the culture medium. We achieved temperature-dependent detachment of cells from Pluronic gels containing or lacking extracellular matrix (ECM) by cooling the gels to 4 degrees C. Using normal human umbilical vein endothelial cells (HUVECs) grown on and released from Pluronic gels lacking ECM, we further found that the expression ratio of the surface markers CD34 and CD105 was twofold higher than for cells grown on polystyrene and removed with trypsin. In addition, the expression ratios for CD34 and CD105 on HUVECs cultivated on the Pluronic gels containing higher concentrations of ECM were lower, which may be due to ECM coating of the cell surface and, thus, interference with antibody binding. In summary, temperature-dependent detachment of cells from Pluronic gels allows the isolation of cells under mild conditions. This can be a powerful tool for surface marker analysis by flow cytometry.     

Macroporous StarPEG-Heparin Cryogels

Macroporous scaffolds with adaptable mechanical and biomolecular properties can be instrumental in enabling cell-based therapies. To meet these requirements, a cryostructuration method was adapted to prepare spongy hydrogels based on chemically cross-linked star-shaped poly(ethylene glycol) (starPEG) and heparin. Subzero temperature treatment of the gel forming reaction mixtures and subsequent lyophilization of the incompletely frozen gels resulted in macroporous biohybrid cryogels showing rapid swelling, porosity of up to 92% with interconnected large pores (30-180 μm), low bulk stiffness, and high mechanical stability upon compression. The applicability of the cryogel scaffolds was investigated using human umbilical vein endothelial cells. Cell attachment and three-dimensional spreading resulted in evenly distributed viable cells within the macroporous starPEG-heparin materials, demonstrating the significant translational potential of the developed three-dimensional cell carriers.     

Reprogramming cardiomyocyte mechanosensing by crosstalk between integrins and hyaluronic acid receptors

The elastic modulus of bioengineered materials has a strong influence on the phenotype of many cells including cardiomyocytes. On polyacrylamide (PAA) gels that are laminated with ligands for integrins, cardiac myocytes develop well organized sarcomeres only when cultured on substrates with elastic moduli in the range 10 kPa-30 kPa, near those of the healthy tissue. On stiffer substrates (>60 kPa) approximating the damaged heart, myocytes form stress fiber-like filament bundles but lack organized sarcomeres or an elongated shape. On soft (<1 kPa) PAA gels myocytes exhibit disorganized actin networks and sarcomeres. However, when the polyacrylamide matrix is replaced by hyaluronic acid (HA) as the gel network to which integrin ligands are attached, robust development of functional neonatal rat ventricular myocytes occurs on gels with elastic moduli of 200 Pa, a stiffness far below that of the neonatal heart and on which myocytes would be amorphous and dysfunctional when cultured on polyacrylamide-based gels. The HA matrix by itself is not adhesive for myocytes, and the myocyte phenotype depends on the type of integrin ligand that is incorporated within the HA gel, with fibronectin, gelatin, or fibrinogen being more effective than collagen I. These results show that HA alters the integrin-dependent stiffness response of cells in vitro and suggests that expression of HA within the extracellular matrix (ECM) in vivo might similarly alter the response of cells that bind the ECM through integrins. The integration of HA with integrin-specific ECM signaling proteins provides a rationale for engineering a new class of soft hybrid hydrogels that can be used in therapeutic strategies to reverse the remodeling of the injured myocardium.     

Sensitization of HT29 colorectal cancer cells to vemurafenib in three‐dimensional collagen cultures

The extracellular matrix to which cancer cells adhere affects cellular sensitivity to anticancer drugs. We sought to examine the changes in sensitivity of colorectal cancer cells carrying the BRAF V600E mutation to vemurafenib cultured in three‐dimensional (3D) collagen‐I gels, while also identifying the signaling pathways involved in these changes. HT29 colorectal cancer cells were cultured in conventional tissue culture (TC) plastic plates or in collagen‐I gels. The HT29 cells demonstrated approximately 10‐fold higher sensitivity to vemurafenib in 3D‐collagen‐I gels compared with those cultured on conventional TC plastic plates. Furthermore, in cells cultured on TC plastic, vemurafenib was found to augment tyrosine phosphorylation of focal adhesion kinase (FAK), while 3D‐cultured cells expressed lower levels of FAK and vemurafenib did not affect its tyrosine phosphorylation, suggesting that FAK contributes to vemurafenib resistance. However, pharmacological inhibition of FAK did not sensitize the cells to vemurafenib. Also, the level of tyrosine‐phosphorylated epidermal growth factor receptor (EGFR)/ERBB2 family proteins was found to be lower in cells cultured in 3D‐collagen gel compared with those in cells cultured on TC plastic. Afatinib, an inhibitor of the EGFR/ERBB family of kinases, sensitized the cells to higher concentrations of vemurafenib, implying their participation in vemurafenib resistance. Adhesion to collagen‐I gel but not to the collagen‐I‐coated plastic surface sensitized the cells, suggesting that the rigidity of the media rather than adherence to collagen‐I may be important for cellular sensitivity to vemurafenib.