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. In the present contribution, we show that embedding human cord blood-derived CD34(+) cells into a collagen I-based hydrogel containing cytokines is a suitable strategy to promote stem cell proliferation and protect these cells from anoxia-induced apoptosis.

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. The versatile method presented allows for the immobilization of biomolecules containing primary amino groups to a broad variety of solid carriers, ranging from silicon oxide surfaces to standard polystyrene well plates and metallic surfaces. The approach can be used to investigate the effects of immobilized enzymes on cell adhesion, and on the catalysis of specific reactions.

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. Surface-immobilized growth factors can be presented over a wide range of concentrations (5-150 ng cm(-2)), as we have previously shown for leukemia inhibitory factor and stem cell factor. Cell activation can be achieved in the presence of adhesion-promoting extracellular matrix proteins. Depending on the methods used, the overall procedure takes 1.5-3 d. In general, the approach can be used to investigate the effect of defined amounts of immobilized growth factors on stem cells and on the maintenance, growth and differentiation of other cell types.

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. High-swelling poly(ethylene-alt-maleic anhydride) (PEMA) copolymer layers permitted significantly higher enzyme loadings and activities than compact poly(octadecene-alt-maleic anhydride) (POMA) films. Substantial fractions of the immobilized, active enzyme layers were found to be conserved upon storage in deionized water for several hours. Ongoing studies explore the potentialities of the developed bioactive coatings to reduce the adhesion of various fouling organisms.

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). Immobilized LIF supported mouse embryonic stem cell (mESC) pluripotency for at least 2 weeks in the absence of added diffusible LIF. Immobilized LIF activated signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) signaling in a dose-dependent manner. The introduced method allows for the robust investigation of cell fate responses from interface-immobilized ligands.

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. The experiments imply a pronounced impact of the prenucleation phase on the cofibril morphology as a result of the strong electrostatic interaction of heparin with tropocollagen. Heparin is assumed to stabilize the collagen microfibrils and to enhance their parallel accretion during cofibrillogenesis with preservation of the typical asymmetric collagen banding pattern. The heparin quantitation data show heparin to be intercalated as a linker molecule with one specific binding site inside the cofibrils. The reconstituted cofibrils with their unusual morphology and GAG intercalation-a phenomenon not reported in vivo-can be expected to exhibit interesting mechanical and biochemical behaviours as a biomaterial for extracellular matrix scaffolds.

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. An increase of the ionic strength with KCl from 10(-4) M to 10(-2) M shifts the isoelectric point (IEP) of the protein from pH 7.5 to 5.3. However, a similar increase of the ionic strength in CaCl(2) solutions shifts the IEP from 7.5 to above pH 9. Enhanced thermal stability with increasing ionic strength was observed by differential scanning calorimetry in both electrolyte systems. In line with this, circular dichroism spectroscopy results show an increase of the helicity with increasing ionic strength. Better screening of charged residues and the formation of salt bridges are assumed to cause the stabilization of collagen I with increasing ionic strength in both electrolyte systems. Preferential adsorption of hydroxide ions onto intrinsically uncharged sites in KCl solutions and calcium binding to negatively charged carboxylic acid moieties in CaCl(2) solutions are concluded to shift the IEP and influence the conformational stability of the protein.

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. After 7 days of culture, the cell number, number of colony-forming units (CFU-C), and gene-expression profile of the cultured cells were assessed. Although the total expansion factor of CD34+ cells was slightly lower when cells were cultivated in the collagen I gel, the frequency of CFU-C was greater than in control suspension cultures. Gene-expression analysis with microarray chip technology revealed the upregulation of more than 50 genes in the presence of collagen I. Among these, genes for several growth factors, cytokines, and chemokines (e.g., IL-8 and macrophage inhibitory protein 1alpha) could be confirmed using quantitative polymerase chain reaction. Furthermore, greater expression levels of the negative cell-cycle regulator BTG2/TIS21 and an inhibitor of the mitogen-activated protein kinase pathway, DUSP2, underline the regulatory role of the extracellular matrix. Together, these data show that the expansion of CD34+ cord blood cells in a culture system containing a three-dimensional collagen I matrix induces a qualitative change in the gene-expression profile of cultivated HSCs.

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. Both inhibitor-containing coatings showed a significantly reduced thrombogenicity, coagulation activation, as well as complement activation when incubated with freshly drawn human whole blood in vitro. The authors conclude that the introduced principle offers a promising approach for hemocompatible materials for short term applications. Even rather simple inhibitors can be successfully employed for that purpose.

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. As a result, the fibronectin conformation is more distorted on the hydrophobic substrates together with remarkably different displacement characteristics in dependence on the adsorbed fibronectin surface concentration and the displacing albumin solution concentration. While the displacement kinetic remains constant for the strongly interacting surface, an acceleration in fibronectin exchange is observed for the weakly interacting surface with increasing fibronectin coverage. For displaced amounts, no change is determined for the hydrophobic substrate, in contrast to the hydrophilic substrate with a decrease of fibronectin exchange with decreasing coverage leading finally to a constant nondisplaceable amount of adsorbed proteins. Furthermore, the variation of the albumin exchange concentration reveals a stronger dependence of the kinetic for the weakly interacting substrate with higher rates at higher albumin concentrations.

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. The free carboxyl surface groups produced by the attachment of polyethylene glycol (PEG) were quantified by Ag(+) labeling and subsequent XPS detection. The film thickness as well as the carboxyl group content were found to be clearly dependent on the copolymer hydrophobicity and the nature of the PEG molecule. For the assessment of the anchorage of the thrombin to the benzamidine-derivative functionalized surfaces, the substrates were immersed in a buffered thrombin solution and the enzyme adsorption was studied using immunostaining/confocal laser scanning microscopy. Higher degrees of thrombin binding were observed for substrates configured with the hydrophilic compared to the more hydrophobic copolymer. Moreover, surface-bound spacers based on alpha,omega-heterobifunctional PEG amino acids (alphaAm,omegaAc-PEG) also enhanced the benzamidine surface density in comparison to homofunctional PEG diacids (alphaAc,omegaAc-PEG) because of a lower degree of carboxyl inactivation due to PEG 'bridging'. Altogether, the choice of copolymer coatings and the type of PEG spacers were demonstrated to enhance the efficiency of the thrombin scavenging by the covalently immobilized coagulation inhibitor.

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. The immobilized collagen assemblies were characterized with respect to the deposited amount of protein using ellipsometry and acidic hydrolysis/HPLC-based amino acid analysis, respectively. AFM, SEM, and cLSM were utilized to gain information on structural features and patterns formed by surface-attached fibrils depending on the initial solution concentrations of collagen. The results revealed that the addition of heparin and hyaluronic acid affected both the fibril dimensions and the meshwork characteristics of the surface-bound fibrils.

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. METHODS: CD34+ HSCs were isolated from cord blood by immunomagnetic separation and cultivated on polymer films coated with reconstituted collagen I fibrils. Cell surface antigens (CD34, CD29) were stained with antibodies, and nuclei were labeled with a DNA stain (TO-PRO-3 iodide) that does not interfere with the fluorochromes of the antibodies. Fluorescence intensity of the adherent cells was measured by means of LSC. Before and after in vitro expansion for time periods of up to 7 days, suspension cells were analyzed with LSC and FCM. RESULTS: LSC-based analysis enabled reliable quantification of CD34+ cells with bright antigen expression before cell culture. At this stage, LSC and FCM data for CD34 expression at given HSC samples largely coincided. After in vitro expansion, LSC data deviated from FCM data for cells with dim CD34 antigen expression, whereas the fluorescence intensity of the CD29 antigen remained comparable. The deviation between LSC and FCM data for CD34dim was attributed to the better resolution of weak fluorescence by FCM. Based on the preceding evaluation of the method, LSC analysis could be applied to characterize HSCs cultivated on collagen I-coated polymer films without detachment of the cells from the substrate. CONCLUSIONS: LSC-based analysis allows for the automated evaluation of adherent HSCs. Although resolution of weakly expressed antigens can be achieved more precisely with FCM, the method provides a valuable tool to study interactions of HSCs with bioartificial substrates.

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.e., in the homodisplacement process) while pure buffer solution and human serum albumin solutions induced desorption or exchange of fibronectin to lower and similar degrees. An increase of the total adsorbed amount of protein due to additional adsorption of fibronectin or human serum albumin accompanied the partial exchange of the preadsorbed fibronectin in the displacement experiments. Evaluation of the kinetics of desorption and exchange of fibronectin at any of the substrates revealed two kinds of surface-attached protein populations--a fast desorbing species and a species with a slow desorption and exchange rate. By a multivariate regression analysis the surface characteristics of the polymer substrate were confirmed to determine the degree of protein desorption and exchange while the dynamics of the layer alteration was found to solely depend on the diffusion behavior of the proteins.

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. Although all of the compared FN coatings were effective in stimulating attachment of endothelial cells, the initial formation of cell-matrix adhesions was found to be controlled by the type of interaction between predeposited FN and the underlying substrate. Covalent linkage and hydrophobic interactions of the predeposited FN with the polymer films interfered with the rapid generation of focal and fibrillar adhesions. It was demonstrated that this was caused by the fact that only weakly bound FN could become readily reorganized by the adherent cells. Upon prolonged culture periods at standard cell culture conditions, secretion and deposition of organized extracellular matrix by the attached cells was found to balance out the differences of the substrates.

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. In addition to the choice of adequate conditions for hydrolysis, derivatization and chromatographic separation extensions of the data evaluation were suggested to improve the accuracy of the approach when applied to single protein systems: comparing the experimentally obtained amino acid ratio to the protein constitution enabled to identify the properly separated and detected amino acids. Those amino acids were selected for a more precise calculation of the amount of immobilized protein. To further increase the accuracy of the method, the retrieval of amino acids corresponding to protein amounts in the range between 0.5 and 4.0 microg was analyzed for a variety of proteins of interest to derive protein-specific correction factors. The evaluation of amino acid data was furthermore applied to quantify binary protein mixtures at similar settings. This method was proven useful to detect the composition of protein mixtures throughout a wide range of absolute and relative concentrations.

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. Examples concern the impact of the substrate-anchorage of fibronectin on the formation of cell-matrix adhesions, the orientation of endothelial cells according to lateral anti-adhesive micropatterns using grafted poly(ethylene oxide), and the spacer-dependent activity of immobilized synthetic thrombin inhibitors.