Poly(ether urethane)s incorporating long alkyl side-chains with terminal carboxyl groups as fatty acid mimics: synthesis, structural characterization and protein adsorption

The object of this work was to produce polyurethanes with greater affinity for albumin (Alb) and improved hemocompatibility by introduction of carboxyl-terminated alkyl side-chains that better mimic fatty acids, in contrast to methyl terminated alkyl side-chains used previously. Synthesis of poly(ether urethane)s (PEUs) with long alkyl side-chains via a multi-step solution addition polymerization is described. The synthesis is based upon the polymerization of a diisocyanate pre-polymer with various chain extenders and reaction with Br-terminated compound in the final stage. The side-chains had terminal methyl or carboxylic groups, and were attached either directly to the polymer backbone or to an oligo(ethylene glycol) spacer. The bulk structure of the PEUs was confirmed by 1H-NMR and the surface polymer structure was characterized by ToF-SIMS. The influence of the incorporated C16-alkyl, C16-carboxyalkyl and oxyethylene-C16-carboxyalkyl side-chains attached to the polymer backbone on fibrinogen (Fg) and Alb adsorption from blood plasma, and Fg adsorption from buffer solutions and binary mixtures with Alb was measured. Incorporation of C16-alkyl or C16-carboxyalkyl side-chains into PEUs caused relatively small changes in Fg and Alb adsorption. PEUs with oxyethylene-C16-carboxyalkyl side-chains exhibited the lowest Fg adsorption and the highest Alb adsorption among all the tested polymers.     

Select bladder smooth muscle cell functions were enhanced on three-dimensional,nano-structured poly(ether urethane) scaffolds

Bladder wall resection is often required as a treatment for invasive bladder cancer. When this happens, a suitable replacement material is needed. The present study, therefore, created three-dimensional, porous, nano-structured poly(ether urethane) (PU) matrices for use as bladder tissue-engineering scaffolds. Select cytocompatibility experiments (specifically adhesion and long-term growth studies) were performed on these scaffolds using human bladder smooth muscle cells (BdSMCs). In addition, the amount of total collagen and elastin present in each cell-seeded scaffold was determined since the production of these extracellular matrix (ECM) proteins is essential for the health and survival of cells and for the functionality of the replaced organ. Finally, to better understand how these scaffolds and resident cells would perform in the complex mechanical environment of the bladder wall, scaffolds and cells were subjected to 10 cmH₂O pressure using a computer-controlled pressure chamber. Results provided evidence that compared to conventionally used, micro-dimensional PU scaffolds, the novel, nanodimensional scaffolds created in this research increased cell adhesion, growth, and ECM protein production. Additionally, scaffolds and resident cells were not affected by exposure to 10 cmH₂O pressure (compared to controls maintained under atmospheric conditions). These results are promising and provide evidence that the nano-dimensional PU scaffolds created in this research are suitable bladder replacement materials that may outperform materials currently used for such purposes.     

Synthesis,Characterization and Cell Compatibility of Novel Poly(ester urethane)s Based on Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Prepared by Melting Polymerization

Novel tailor-made poly(ester urethane)s (PUs) based on microbial polyesters poly(3-hydroxybutyrate-co-4hydroxybutyrate) (P3HB4HB) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) were synthesized by melting polymerization (MP) using 1,6-hexamethylene diisocyanate (HDI) as a coupling agent. A comprehensive characterization using ¹H-NMR, Fourier transform infrared spectroscopy (FT-IR), gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), mechanical properties, static water contact angles, cell proliferation using smooth muscle cells from rabbit aorta (RaSMCs) and immortalized human keratinocytes (HaCat), and blood coagulation behavior were conducted on the synthesized PUs films. DSC showed that PU samples had a low degree of crystallinity at room temperature and became fully amorphous after a melt-quenched process. The series of tailor-made PUs based on different mass ratios of P3HB4HB and PHBHHx revealed a ductile and flexile mechanical property especially for PHBHHx-rich PU, or a hydrophobic property for 4HB-rich PU. A 4 days incubation experiment showed that all PU films had a better cell proliferation than poly(lactic acid) (PLA), polyhydroxybutyrate (PHB), P3HB4HB and PHBHHx. RaSMCs cultured on PU films had a quiescent contractile phenotype, indicating that they were fully functional. HaCat incubated on tailor-made PU films showed a proliferation approximately equal to tissue-culture plates (TCPs). Blood coagulation behavior tests revealed a strong platelet adhesion and a short coagulation time on PU films. This study demonstrated potential medical applications for P3HB4HB and PHBHHx based polyurethane as a hydrophobic wound-healing and hemostatic materials.     

Poly(acrylic acid)-grafted Poly(N-isopropyl acrylamide) Networks: Preparation,Characterization and Hydrogel Behavior

Poly(acrylic acid)-grafted poly(N-isopropylacrylamide) co-polymer networks (PNIPAAm-g-PAA) were prepared via the reversible addition-fragmentation transfer (RAFT) polymerization of N-isopropyl- acrylamide (NIPAAm) with trithiocarbonate-terminated PAA as a macromolecular chain-transfer agent in the presence of N,N-methylenebisacrylamide. The PNIPAAm-g-PAA co-polymer networks were characterized by means of Fourier transform infrared spectroscopy, differential scanning calorimetry and small-angle X-ray scattering. It is found that the PNIPAAm-g-PAA co-polymer networks were microphase-separated, in which the microdomains of PNIPAAm-PAA interpolymer complexes were dispersed into the PNIPAAm matrix. The PNIPAAm-g-PAA hydrogels displayed a dual response to temperature and pH values. The thermoresponsive properties of PNIPAAm-g-PAA networks were investigated. Below the volume phase transition temperatures, the PNIPAAm-g-PAA hydrogels possessed much higher swelling ratios than control PNIPAAm hydrogel. In terms of swelling, deswelling and reswelling tests, it is judged that the PNIPAAm-g-PAA hydrogels displayed faster response to the external temperature changes than control PNIPAAm hydrogel. The improved thermoresponsive properties of hydrogels are ascribed to the formation of PAA-grafted PNIPAAm networks, in which the water-soluble PAA chains behave as the hydrophiphilic tunnels and allow water molecules to go through and, thus, to accelerate the diffusion of water molecules.     

Poly(ether imide) membranes: studies on the effect of surface modification and protein pre-adsorption on endothelial cell adhesion,growth and function

Poly(ether imide) (PEI) membranes of which the surface was modified with carboxylic groups were tested in comparison to pure PEI and poly(ethylene terephtalate) (PET) for their ability to support attachment, growth and function of human umbilical vein endothelial cells (HUVEC) with respect to endothelization of the above materials. Flat sheet PEI membranes were modified by covalent binding of iminodiacetic acid (IDA) for different periods of time (1 to 30 min) to obtain surfaces with various content of carboxylic groups. In addition, fibronectin (FN) and fibrinogen (FNG) pre-adsorption on the various membranes were studied for their effect on HUVEC behaviour. The results show a decreased protein adsorption and HUVEC adhesion, growth and function in terms of prostacyclin production with an increase in carboxylic groups. Pre-adsorption of the membranes with FN or FNG promoted activity of HUVEC, which became superior to cells on PET. FN-coated membranes were found to be a better substrate for HUVEC adhesion and prostacyclin production, while on FNG-coated membranes cells grew better. Overall it can be concluded that PEI is a promising materials for endothelial cells immobilization as it is needed for improving the haemocompatibility of cardiovascular devices.     

Amoxicillin-Loaded Sponges Made of Collagen and Poly[(methyl vinyl ether)-co-(maleic anhydride)] for Root Canal Treatment: Preparation,Characterization and In Vitro Cell Compatibility

The difficulty of eliminating Enterococcus faecalis and other bacteria infecting dental root canals makes it desirable to develop formulations capable of sustained release of antibiotics within the canal. With this function in view, in this work we compared the mechanical, drug release and biocompatibility properties of amoxicillin-loaded collagen (CL) and CL complexed with poly[(methyl vinyl ether)-co-(maleic anhydride)] (PVMMA), with or without glutaraldehyde (GTA) or the natural product genipin (GN) as cross-linker. Collagen was not denatured by complexation with PVMMA. Only CL–PVMMA–GN sponges did not disintegrate during 7 days exposure to cell culture medium (un-cross-linked CL disintegrated within 24 h and un-cross-linked CL–PVMMA within 4 days), and CL–PVMMA–GN sponges also exhibited the most appropriate combination of mechanical properties (hardness, modulus of deformability and plasticity). CL–PVMMA–GN sponges absorbed aqueous medium faster than other cross-linked formulations, but their maximum uptake was less; and drug release from CL–PVMMA–GN sponges tended to be faster than from any other, except un-cross-linked CL–PVMMA, maximum release taking about 4 days. No formulation significantly altered the viability of L929 fibroblast-like mouse connective tissue cells, but cells growing on sponges showed signs of non-adherence. It is concluded that genipin-cross-linked CL–PVMMA sponges merit further investigation as antibiotics vehicles and aids to tissue regeneration in the dental root canal.     

Chitosan-poly(acrylic acid) polyelectrolyte complex membranes: preparation,characterization and permeability studies

Polyelectrolyte complex (PEC) membranes were obtained by mixing solutions of two polymers of opposite charges, chitosan (Chi) and poly(acrylic acid) PAA. Three membranes were obtained: one made of pure chitosan and two membranes with chitosan mixed with PAA at a ratio of 95:5 (one prepared using PAA solution in 3.5% formic acid, named ChiPAA3.5, and another one using a PAA solution in 10% formic acid, named ChiPAA10). The membranes were characterized by swelling experiments, FT-IR spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), mechanical properties and permeability studies in relation to a drug model (sodium sulphamerazine). The calculation of degree of ionization showed that the lower the formic acid concentration was, the higher the PAA dissociation degree. Polyelectrolyte complex formation was characterized by FT-IR. Water uptake results showed that PEC membranes were more hydrophilic than pure chitosan, ChiPAA3.5 being the most. Morphological analysis by SEM and AFM showed that PAA addition changed the membranes morphology, especially for ChiPAA3.5. Mechanical properties indicated that PEC membranes were more rigid than pure chitosan membranes and that the morphology has an influence on tensile strength values. Permeability values decreased with complex formation and were lower for ChiPAA10 than ChiPAA3.5. However, as drug concentration was increased, the difference between the two complex membranes disappeared. The results were discussed considering the drug–membrane interactions. Diffusion coefficient values indicated that ChiPAA3.5 had a higher drug retention capacity than ChiPAA10.     

Protein adsorption to oligo(ethylene glycol) self-assembled monolayers: Experiments with fibrinogen,heparinized plasma,and serum

Low protein adsorption is believed advantageous for blood-contacting materials and ethylene glycols (EG)-based polymeric compounds are often attached to surfaces for this purpose. In the present study, the adsorption of fibrinogen, serum, and plasma were studied by ellipsometry on a series of well-defined oligo(EG) terminated alkane-thiols self-assembled on gold. The layers were prepared with compounds of the general structure HS-(CH₂)₁₅-CONH-EG_n, where n = 2, 4, and 6. Methoxy-terminated tri(EG) undecanethiol and hydroxyl-terminated hexadecanethiol self-assembled monolayers (SAMs) were used as references. The results clearly demonstrate that the adsorption depends on the experimental conditions with small amounts of fibrinogen adsorbing from a single protein solution, but larger amounts of proteins from serum and plasma. The adsorption of fibrinogen and blood plasma decreased with an increasing number of EG repeats and was temperature-dependent. Significantly less serum adsorbed to methoxy tri(EG) than to hexa(EG) and more proteins remained on the latter surface after incubation in a sodium dodecyl sulfate (SDS) solution, indicating a looser protein binding to the methoxy-terminated surface. All surfaces adsorbed complement factor 3 (C3) from serum and plasma, although no surfacemediated complement activation was observed. The present study points to the importance of a careful choice of the protein model system before general statements regarding the protein repellant properties of potential surfaces can be made.     

Comparison of Bone Marrow Stromal Cell Behaviors on Poly(caprolactone) with or without Surface Modification: Studies on Cell Adhesion,Survival and Proliferation

Poly(caprolactone) (PCL) is a promising biodegradable polymer for tissue engineering. However, intrinsically poor cell-adhesive properties of PCL may limit its application. In this study, the PCL film surface was modified with RGDC peptide by a chemical immobilization procedure. Furthermore, bone marrow stromal cell (BMSC) behaviors including attachment, spreading, focal adhesion formation, focal adhesion kinase (FAK) activation, apoptosis and proliferation when cultured on the modified PCL films were investigated. Our results demonstrated that PCL with RGD modification promoted initial BMSC attachment, spreading and focal adhesion formation. At a later time point (12 h), BMSC attachment on both RGD peptide-modified PCL and PCL-NH₂ films significantly increased compared to untreated PCL films. Importantly, FAK phosphorylation was significantly increased only on the films with RGD-modified films, not on the PCL-NH₂ films, demonstrating that PCL with RGD modification had an advantage in initiating the specific integrin-mediated signal transduction and might play an important role in the subsequent retardation in cell death and enhancement in cell proliferation. The present results provide more evidence that functionalizing PCL with RGD peptides may be a feasible way to improve the interaction between BMSC and PCL substrate, which is important in tissue engineering.     

Protein interactions with oligo(ethylene glycol) (OEG) self-assembled monolayers: OEG stability,surface packing density and protein adsorption

We present a study of protein adsorption on oligo(ethylene glycol) (OEG) self-assembled monolayers (SAMs) at a range of OEG surface densities. OEG SAMs were formed in mixed ethanol and water solutions at different assembly temperatures to adjust the packing density of EG₄-SAMs. These SAMs were characterized using X-ray photoelectron spectroscopy (XPS). Fibrinogen adsorption on these surfaces was measured by a surface plasmon resonance (SPR) sensor at different temperatures. This work is aimed at addressing three important issues for protein–OEG interactions, i.e., (i) OEG stability, (ii) the correlation between OEG surface densities and surface non-fouling properties, and (iii) protein adsorption on OEG surfaces at different temperatures.     

Enzyme-linked immunosorbent assay (ELISA) with covalently bound protein on glass tubes: 1. Stable antigenicity and binding of IgG as a model antigen after repeated use

A modification of the ELISA procedure is described. The system is based on the covalent binding of protein to glass tubes. Human IgG was used as model antigen. Optimal conditions were tested for the removal of alkaline phosphatase-labeled antibodies from their antigen. Under such optimal conditions, a regenerable system could be created which exhibited many advantages, as compared with conventional ELISAs with antigens absorbed unspecifically to plastic surfaces. The advantages are: 1. A higher density of IgG as model antigen on the solid phase, i.e., 1 molecule IgG per 94 nm2 with glass tubes as compared with 110 nm2 with polystyrene (PS) or 143 nm2 with polyvinylchloride (PVC) microtiter plates. 2. A much smaller unspecific absorption of less than 1% as compared with 39% with PS-plates or 16% with PVC-plates treated under identical conditions. 3. A higher stability of the binding of the model antigen to the solid phase, i.e., a drastically reduced protein loss of less than 6% during the first ELISA procedure (including the regeneration) and of less than 1% during the subsequent ELISA and regeneration cycles with glass tubes as compared with 46% (PS plates) or 55% (PVC plates). 4. A smaller intraday variation coefficient of the ELISAs of 4.8% with glass tubes as opposed to 9.7% or 7.5% with PS or PVC plates respectively. The system with covalently bound antigens on glass tubes could be used in at least 20 consecutive measuring cycles. In five consecutive cycles, a protein loss of less than 5% was observed and the interday variation coefficient of the ELISA reaction was smaller than 5% using the same tubes repeatedly. Our results indicate that covalent linkage of protein can improve ELISA and lead to repeatedly usable systems as long as the antigen is stable against the regeneration procedure. Such an ELISA system may be helpful with highly purified proteins.