Growth induction and low-oxygen apoptosis inhibition of human CD34+ progenitors in collagen gels.

Various reports have indicated low survival of injected progenitors into unfavorable environments such as the ischemic myocardium or lower limb tissues. This represents a major bottleneck in stem-cell-based cardiovascular regenerative medicine. Strategies to enhance survival of these cells in recipient tissues have been therefore sought to improve stem cell survival and ensure long-term engraftment.

Enzyme immobilization on reactive polymer films.

Immobilized enzymes are currently used in many bioanalytical and biomedical applications. This protocol describes the use of thin films of maleic anhydride copolymers to covalently attach enzymes directly to solid supports at defined concentrations. The concentration and activity of the surface-bound enzymes can be tuned over a wide range by adjusting the concentration of enzyme used for immobilization and the physicochemical properties of the polymer platform, as demonstrated here for the proteolytic enzyme Subtilisin A.

Immobilization of growth factors on solid supports for the modulation of stem cell fate.

Surface- and matrix-bound signals modulate stem cell fate in vivo and in vitro. This protocol enables the immobilization of a wide range of biomolecules that contain primary amino groups to different types of solid carriers, including glass substrates and standard polystyrene well plates. We describe how thin polymer coatings of poly(octadecene-alt-maleic anhydride) can be used to covalently attach growth factors directly, or through poly(ethylene glycol) spacers, to solid supports at defined concentrations.

Covalent immobilization of Subtilisin A onto thin films of maleic anhydride copolymers.

Enzymes cleaving the biopolymer adhesives of fouling organisms are attracting attention for the prevention of biofouling. We report a versatile and robust method to confine the serine protease Subtilisin A (or Subtilisin Carlsberg) to surfaces to be protected against biofouling. The approach consists of the covalent immobilization of the protease onto maleic anhydride copolymer thin film coatings.

Functional immobilization of signaling proteins enables control of stem cell fate.

The mode of ligand presentation has a fundamental role in organizing cell fate throughout development. We report a rapid and simple approach for immobilizing signaling ligands to maleic anhydride copolymer thin-film coatings, enabling stable signaling ligand presentation at interfaces at defined concentrations. We demonstrate the utility of this platform technology using leukemia inhibitory factor (LIF) and stem cell factor (SCF).

Heparin intercalation into reconstituted collagen I fibrils: Impact on growth kinetics and morphology.

Collagen type I fibrils, reconstituted in vitro in the presence of heparin, exhibit an unusually thick and straight shape. A detailed structural analysis by scanning force and scanning electron microscopy revealed a non-linear dependence in size distribution, width-to-length ratio, and morphology over a wide range of glycosaminoglycan (GAG) concentrations. By varying molecular weight, degree of sulphation, charge, and concentration of different GAGs we are able to correlate the morphological data with kinetic turbidimetric measurements, and quantitation of fibril-bound GAG.

Electrostatic interactions modulate the conformation of collagen I.

The pH- and electrolyte-dependent charging of collagen I fibrils was analyzed by streaming potential/streaming current experiments using the Microslit Electrokinetic Setup. Differential scanning calorimetry and circular dichroism spectroscopy were applied in similar electrolyte solutions to characterize the influence of electrostatic interactions on the conformational stability of the protein. The acid base behavior of collagen I was found to be strongly influenced by the ionic strength in KCl as well as in CaCl(2) solutions.

Benzamidine-based coatings: Implication of inhibitor structure on the inhibition of coagulation enzymes in solution and in vitro hemocompatibility assessment.

Synthetic inhibitors of trypsin-like serine proteases were covalently immobilized to polymeric materials to passivate coagulation enzymes during blood contact. The inhibitory potency of a structurally simple and larger, more complex amidine derivatives was assessed against thrombin and factor Xa. After adsorption of serum albumin, the polymer films decorated with either one of the inhibitors were found to scavenge thrombin-with a higher affinity in the case of the larger inhibitor-but not factor Xa.

Immobilization of an anticoagulant benzamidine derivative: effect of spacer arms and carrier hydrophobicity on thrombin binding.

Prevention of blood coagulation is very often a prerequisite for successful medical devices. For that purpose, passivation of the key coagulation enzyme thrombin through the derivatization of the material's surface with an amidine-based molecule has been found to be promising. To further enhance the efficiency of this approach, thin layers of maleic anhydride copolymers offering different physico-chemical characteristics were tethered with carboxyl terminated polyethylene glycol to covalently immobilize a benzamidine-type derivative.

Gene-expression profiling of CD34+ hematopoietic cells expanded in a collagen I matrix.

CD34+ hematopoietic stem/progenitor cells (HSCs) reside in the bone marrow in close proximity to the endosteal bone surface, surrounded by osteoblasts, stromal cells, and various extracellular matrix molecules. We used a bioartificial matrix of fibrillar collagen I, the major matrix component of bone, as a scaffold for ex vivo expansion of HSCs. CD34+ HSCs were isolated from umbilical cord blood and cultivated within reconstituted collagen I fibrils in the presence of fms-like tyrosine kinase-3 ligand, stem cell factor, and interleukin (IL)-3.

Fibronectin displacement at polymer surfaces.

The interactions of fibronectin with thin polymer films are studied in displacement experiments using human serum albumin. Fibronectin adsorption and exchange on two different maleic anhydride copolymer surfaces differing in hydrophobicity and surface charge density have been analyzed by quartz crystal microbalance and laser scanning microscopy with respect to adsorbed amounts, viscoelastic properties, and conformation. Fibronectin is concluded to become attached onto hydrophilic surfaces as a "softer", less rigid protein layer, in contrast to the more rigid, densely packed layer on hydrophobic surfaces.

Dynamic alterations of fibronectin layers on copolymer substrates with graded physicochemical characteristics.

Desorption and exchange of preadsorbed fibronectin layers in pure buffer solution and solutions of human serum albumin or fibronectin, respectively, were studied in dependence on the physicochemical characteristics of maleic acid copolymer films used as substrates. Although the preadsorbed amount of fibronectin differed only slightly, the protein was found to exhibit a significantly enhanced anchorage at the more hydrophobic polymer surface as compared to the more hydrophilic and more negatively charged polymer surface. The preadsorbed fibronectin layer was most efficiently exchanged by fibronectin (i.

In vitro reconstitution of fibrillar collagen type I assemblies at reactive polymer surfaces.

The reconstitution of fibrillar collagen and its assemblies with heparin and hyaluronic acid was studied in vitro. Fibril formation kinetics were analyzed by turbidity and depletion measurements in solutions containing varied concentrations of collagen and glycosaminoglycans. Fibril-forming collagen solutions were further applied for the coating of planar substrates which had been modified with alternating maleic anhydride copolymer films before.

Comparison of flow cytometry and laser scanning cytometry for the analysis of CD34+ hematopoietic stem cells.

Background: Characterization of hematopoietic stem cells (HSCs) by laser scanning cytometry (LSC) was compared with conventional flow cytometry (FCM). The method was evaluated for application in the development of advanced cell culture substrates that were supposed to support the ex vivo expansion of HSC. For this purpose, adherent HSCs were grown in culture on thin polymer films coated with reconstituted collagen I fibrils and subsequently analyzed by LSC.

Fibronectin anchorage to polymer substrates controls the initial phase of endothelial cell adhesion.

Early stages of the adhesion of human endothelial cells onto a set of smooth polymer films were analyzed to reveal the modulation of cell-matrix interactions by the physicochemical constraints of predeposited fibronectin (FN). Hydrophobic and hydrophilic polymer substrates, consisting of poly(octadecene-alt-maleic anhydride) and poly(propene-alt-maleic anhydride) films, were coated with similar amounts of FN at conditions of either covalent or noncovalent immobilization. The well-defined substrates permit variation of the anchorage of FN at invariant topography, pliability, and molecular composition.

Quantitative analysis of immobilized proteins and protein mixtures by amino acid analysis.

Biomolecular surface engineering of materials often requires precise, versatile and efficient quantification of immobilized proteins at solid surfaces. Acidic hydrolysis of surface-bound proteins and subsequent HPLC analysis of fluorescence-derivatized amino acids were adapted and critically evaluated for that purpose. Contaminations and concentration-dependent amino acid retrieval during HPLC were found to influence the accuracy of the method.

Maleic anhydride copolymers--a versatile platform for molecular biosurface engineering.

A platform of thin polymer coatings was introduced for the functional modulation of immobilized bioactive molecules at solid/liquid interfaces. The approach is based on covalently attached alternating maleic acid anhydride copolymers with a variety of comonomers and extended through conversion of the anhydride moieties by hydrolysis, reaction with functional amines, and other conversions of the anhydride moieties. We demonstrate that these options permit control of the physicochemical constraints for bioactive molecules immobilized at interfaces to influence important performance characteristics of biofunctionalized materials for medical devices and molecular diagnostics.