Characterization of Blow-Spun Polyurethane Scaffolds-Influence of Fiber Alignment and Fiber Diameter on Pericyte Growth.

In this study, fibrous polyurethane (PU) materials with average fiber diameter of 200, 500, and 1000 nm were produced using a solution blow spinning (SBS) process. The effects of the rotation speed of the collector (in the range of 200-25 000 rpm) on the fiber alignment and diameter were investigated. The results showed that fiber alignment was influenced by the rotation speed of the collector, and such alignment was possible when the fiber diameter was within a specific range. Homogeneously oriented fibers were obtained only for a fiber diameter ≥500 nm. Moreover, the changes in fiber orientation and fiber diameter (resulting from changes in the rotation speed of the collector) were more noticeable for materials with an average fiber diameter of 1000 nm in comparison to 500 nm, which suggests that the larger the fiber diameter, the better the controlled architectures that can be obtained. The porosity of the produced scaffolds was about 65-70%, except for materials with a fiber diameter of 1000 nm and aligned fibers, which had a higher porosity (76%). Thus, the scaffold pore size increased with increasing fiber diameter but decreased with increasing fiber alignment. The mechanical properties of fibrous materials strongly depend on the direction of stretching, whereby the fiber orientation influences the mechanical strength only for materials with a fiber diameter of 1000 nm. Furthermore, the fiber diameter and alignment affected the pericyte growth. Significant differences in cell growth were observed after 7 days of cell culture between materials with a fiber diameter of 1000 nm (cell coverage 96-99%) and those with a fiber diameter of 500 nm (cell coverage 70-90%). By appropriately setting the SBS process parameters, scaffolds can be easily adapted to the cell requirements, which is of great importance in producing complex 3D structures for guided tissue regeneration.

Multilayered blow-spun vascular prostheses with luminal surfaces in Nano/Micro range: the influence on endothelial cell and platelet adhesion.

BACKGROUND: In this study, two types of polyurethane-based cylindrical multilayered grafts with internal diameters ≤ 6 mm were produced by the solution blow spinning (SBS) method. The main aim was to create layered-wall prostheses differing in their luminal surface morphology. Changing the SBS process parameters, i.e. working distance, rotational speed, volume, and concentration of the polymer solution allowed to obtain structures with the required morphologies. The first type of prostheses, termed Nano, possessed nanofibrous luminal surface, and the second type, Micro, presented morphologically diverse luminal surface, with both solid and microfibrous areas. RESULTS: The results of mechanical tests confirmed that designed prostheses had high flexibility (Young's modulus value of about 2.5 MPa) and good tensile strength (maximum axial load value of about 60 N), which meet the requirements for vascular prostheses. The influence of the luminal surface morphology on platelet adhesion and the attachment of endothelial cells was investigated. Both surfaces did not cause hemolysis in contact with blood, the percentage of platelet-occupied area for Nano and Micro surfaces was comparable to reference polytetrafluoroethylene (PTFE) surface. However, the change in morphology of surface-adhered platelets between Nano and Micro surfaces was visible, which might suggest differences in their activation level. Endothelial coverage after 1, 3, and 7 days of culture on flat samples (2D model) was higher on Nano prostheses as compared with Micro scaffolds. However, this effect was not seen in 3D culture, where cylindrical prostheses were colonized using magnetic seeding method. CONCLUSIONS: We conclude the produced scaffolds meet the material and mechanical requirements for vascular prostheses. However, changing the morphology without changing the chemical modification of the luminal surface is not sufficient to achieve the appropriate effectiveness of endothelialization in the 3D model.

The effect of surface morphology on endothelial and smooth muscle cells growth on blow-spun fibrous scaffolds.

This study aimed to analyze the growth of two types of blood vessel building cells: endothelial cells (ECs) and smooth muscle cells (SMCs) on surfaces with different morphology. Two types of materials, differing in morphology, were produced by the solution blow spinning technique. One-layer materials consisted of one fibrous layer with two fibrous surfaces. Bi-layer materials consisted of one fibrous-solid layer and one fibrous layer, resulting in two different surfaces. Additionally, materials with different average fiber diameters (about 200, 500, and 900 nm) were produced for each group. It has been shown that it is possible to obtain structures with a given morphology by changing the selected process parameters (working distance and polymer solution concentration). Both morphology (solid versus fibrous) and average fiber diameter (submicron fibers versus microfibers) of scaffolds influenced the growth of ECs. However, this effect was only visible after an extended period of culture (6 days). In the case of SMCs, it was proved that the best growth of SMCs is obtained for micron fibers (with an average diameter close to 900 nm) compared to the submicron fibers (with an average diameter below 900 nm).

Vascular Polyurethane Prostheses Modified with a Bioactive Coating-Physicochemical, Mechanical and Biological Properties.

This study describes a method for the modification of polyurethane small-diameter (5 mm) vascular prostheses obtained with the phase inversion method. The modification process involves two steps: the introduction of a linker (acrylic acid) and a peptide (REDV and YIGSR). FTIR and XPS analysis confirmed the process of chemical modification. The obtained prostheses had a porosity of approx. 60%, Young's Modulus in the range of 9-11 MPa, and a water contact angle around 40°. Endothelial (EC) and smooth muscle (SMC) cell co-culture showed that the surfaces modified with peptides increase the adhesion of ECs. At the same time, SMCs adhesion was low both on unmodified and peptide-modified surfaces. Analysis of blood-materials interaction showed high hemocompatibility of obtained materials. The whole blood clotting time assay showed differences in the amount of free hemoglobin present in blood contacted with different materials. It can be concluded that the peptide coating increased the hemocompatibility of the surface by increasing ECs adhesion and, at the same time, decreasing platelet adhesion. When comparing both types of peptide coatings, more promising results were obtained for the surfaces coated with the YISGR than REDV-coated prostheses.

Effect of Extreme Ultraviolet (EUV) Radiation and EUV Induced, N2 and O2 Based Plasmas on a PEEK Surface's Physico-Chemical Properties and MG63 Cell Adhesion.

Polyetheretherketone (PEEK), due to its excellent mechanical and physico-chemical parameters, is an attractive substitute for hard tissues in orthopedic applications. However, PEEK is hydrophobic and lacks surface-active functional groups promoting cell adhesion. Therefore, the PEEK surface must be modified in order to improve its cytocompatibility. In this work, extreme ultraviolet (EUV) radiation and two low-temperature, EUV induced, oxygen and nitrogen plasmas were used for surface modification of polyetheretherketone. Polymer samples were irradiated with 100, 150, and 200 pulses at a 10 Hz repetition rate. The physical and chemical properties of EUV and plasma modified PEEK surfaces, such as changes of the surface topography, chemical composition, and wettability, were examined using atomic force microscopy (AFM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and goniometry. The human osteoblast-like MG63 cells were used for the analysis of cell viability and cell adhesion on all modified PEEK surfaces. EUV radiation and two types of plasma treatment led to significant changes in surface topography of PEEK, increasing surface roughness and formation of conical structures. Additionally, significant changes in the chemical composition were found and were manifested with the appearance of new functional groups, incorporation of nitrogen atoms up to ~12.3 at.% (when modified in the presence of nitrogen), and doubling the oxygen content up to ~25.7 at.% (when modified in the presence of oxygen), compared to non-modified PEEK. All chemically and physically changed surfaces demonstrated cyto-compatible and non-cytotoxic properties, an enhancement of MG63 cell adhesion was also observed.

Cylindrical Polyurethane Scaffold Fabricated Using the Phase Inversion Method: Influence of Process Parameters on Scaffolds' Morphology and Mechanical Properties.

This work presents a method of obtaining cylindrical polymer structures with a given diameter (approx. 5 mm) using the phase inversion technique. As part of the work, the influence of process parameters (polymer hardness, polymer solution concentration, the composition of the non-solvent solution, process time) on the scaffolds' morphology was investigated. Additionally, the influence of the addition of porogen on the scaffold's mechanical properties was analyzed. It has been shown that the use of a 20% polymer solution of medium hardness (ChronoFlex C45D) and carrying out the process for 24 h in 0:100 water/ethanol leads to the achievement of repeatable structures with adequate flexibility. Among the three types of porogens tested (NaCl, hexane, polyvinyl alcohol), the most favorable results were obtained for 10% polyvinyl alcohol (PVA). The addition of PVA increases the range of pore diameters and the value of the mean pore diameter (9.6 ± 3.2 vs. 15.2 ± 6.4) while reducing the elasticity of the structure (Young modulus = 3.6 ± 1.5 MPa vs. 9.7 ± 4.3 MPa).

Surface Modification of PLLA, PTFE and PVDF with Extreme Ultraviolet (EUV) to Enhance Cell Adhesion.

Recently, extreme ultraviolet (EUV) radiation has been increasingly used to modify polymers. Properties such as the extremely short absorption lengths in polymers and the very strong interaction of EUV photons with materials may play a key role in achieving new biomaterials. The purpose of the study was to examine the impact of EUV radiation on cell adhesion to the surface of modified polymers that are widely used in medicine: poly(tetrafluoroethylene) (PTFE), poly (vinylidene fluoride) (PVDF), and poly-L-(lactic acid) (PLLA). After EUV surface modification, which has been performed using a home-made laboratory system, changes in surface wettability, morphology, chemical composition and cell adhesion polymers were analyzed. For each of the three polymers, the EUV radiation differently effects the process of endothelial cell adhesion, dependent of the parameters applied in the modification process. In the case of PVDF and PTFE, higher cell number and cellular coverage were obtained after EUV radiation with oxygen. In the case of PLLA, better results were obtained for EUV modification with nitrogen. For all three polymers tested, significant improvements in endothelial cell adhesion after EUV modification have been demonstrated.

Evaluation of Sterilization/Disinfection Methods of Fibrous Polyurethane Scaffolds Designed for Tissue Engineering Applications.

Sterilization of a material carries the risk of unwanted changes in physical and chemical structure. The choice of method is a challenge-the process must be efficient, without significantly changing the properties of the material. In the presented studies, we analyzed the effect of selected sterilization/disinfection techniques on the properties of nanofibrous polyurethane biomaterial. Both radiation techniques (UV, gamma, e-beam) and 20 minutes' contact with 70% EtOH were shown not to achieve 100% sterilization efficiency. The agar diffusion test showed higher sterilization efficiency when using an antimicrobial solution (AMS). At the same time, none of the analyzed techniques significantly altered the morphology and distribution of fiber diameters. EtOH and e-beam sterilization resulted in a significant reduction in material porosity together with an increase in the Young's modulus. Similarly, AMS sterilization increased the value of Young's modulus. In most cases, the viability of cells cultured in contact with the sterilized materials was not affected by the sterilization process. Only for UV sterilization, cell viability was significantly lower and reached about 70% of control after 72 h of culture.

Fenton-type reaction grafting of polyvinylpyrrolidone onto polypropylene membrane for improving hemo- and biocompatibility.

Known techniques for modification of polypropylene membranes (PPm) often require modification of the membrane in its entire volume (i.e. at the manufacturing stage), which may affect its properties. In the present work, the authors proposed a simple method for PPm hydrophilization. The process involves a two-step Fenton-type reaction, with ethylene glycol dimethacrylate (EGDMA) as a crosslinking agent and cumene hydroperoxide (CHP) as a source of free radicals. This hydrogel coating aims to enhance membrane hemocompatible and biocompatible properties. The biggest advantage of the proposed technique is the change of materials' surface properties, without interfering with its internal structure. Microscopic (SEM) and spectroscopic (FTIR-ATR) analyses confirmed the presence of hydrogel coating on PPm surfaces. Additionally, the evaluation of the surface density of the coating showed that the thickness of the coating increases with the reaction time and CHP concentration. The applied coatings significantly increase surface hydrophilicity (contact angle for PPm: 128.58°â€¯±â€¯0.52°, for all modified surfaces <53.31°â€¯±â€¯2.03°). The cytotoxicity test (XTT assay) proved biocompatibility of the PVP coating - cell viability remained above 90% for all variants tested. The modification resulted in a decrease in fibrinogen adsorption (of at least about 16%) and in a number of surface-adhered platelets. The assay evaluating the amount of secreted cell adhesion molecules (ICAM-1) showed a significant reduction (of at least about 50%) in the expression of ICAM-1 for all hydrogel-modified surfaces.

Polyvinylpyrrolidone (PVP) hydrogel coating for cylindrical polyurethane scaffolds.

The presented study describes a method for the preparation and modification of cylindrical polyurethane structures with polyvinylpyrrolidone (PVP) hydrogel coating. The modified polyurethane scaffolds were fabricated using the phase-inversion technique and intended to be used as a vascular prosthesis. The proposed modification method involves a two-step Fenton-type reaction. Physicochemical analysis (Fourier transform infrared spectroscopy, scanning electron microscopy) confirmed the presence of the hydrogel coating. The influence of PVP and polymerization initiator (cumene hydroperoxide) concentrations on hydrogel's properties were examined. The higher concentrations of reagent were used, the thicker coating was obtained. After modification, the material's surface becomes more hydrophilic in comparison to pristine polyurethane. Cytotoxicity assay (MTT test) confirmed that PVP-coating is not toxic. The introduction of hydrogel coating resulted in a significant decrease in the fibrinogen adsorbed to the material's surface as compared to a non-modified polymer. Platelet adhesion assay demonstrated almost no platelet adhesion to the modified surfaces.

Determination of polyurethane-grafted peptide (GSGREDVGSG) using bicinchoninic acid assay.

The goal in the presented study was to develop a simple, fast and accurate method for measuring the surface density of a short peptide sequence bound to a polymeric substrate. We analyzed polyurethane samples chemically modified with acrylic acid and polyurethane-grafted peptide (GSGREDVGSG) and investigated the possibility of using the bicinchoninic acid (BCA) assay to determine surface density of the solid-supported peptide. We set the conditions (temperature, time) under which the test should be conducted. We also studied the interaction of the BCA reagent with polyurethane substrate and the effect of drying conditions as well as material type and form on the test response. We have proposed potential factors that might interfere with the BCA assay and chosen the proper control materials.

Endothelialization of polyurethanes: Surface silanization and immobilization of REDV peptide.

The paper presents method for chemical immobilization of arginine-glutamic acid-aspartic acid-valine (REDV) peptide on polyurethane surface. The peptide has been covalently bonded using silanes as a spacer molecules. The aim of this work was to investigate the proposed modification process and assess its biological effectiveness, especially in contact with blood and endothelial cells. Physicochemical properties were examined in terms of wettability, atomic composition and density of introduced functional groups and peptide molecules. Experiments with blood showed that material coating reduced number of surface-adhered platelets and fibrinogen molecules. In contrast to polyurethane (PU), there were no blood components deposited on REDV-modified materials (PU-REDV); fibrinogen adsorption on PU-REDV surface has been strongly reduced compared to PU. Analysis of cell adhesion after 1, 2, 3, 4, and 5 days of culture showed a significant increase of the cell-coated area on PU-REDV compared to PU. However, an intense cell growth appeared also on the control surface modified without the addition of REDV. Thus, the positive effect of REDV peptide on the adhesion of HUVEC could not be unequivocally confirmed.

Athrombogenic hydrogel coatings for medical devices--Examination of biological properties.

In the article the authors present hydrogel coatings prepared from polyvinylpyrrolidone (PVP) macromolecules, which are chemically bonded to polyurethane (PU) substrate. The coating is designed to improve the surface hemocompatibility of blood-contacting medical devices. The coating was characterized in terms of physical properties (swelling ratio, hydrogel density, surface morphology, coating thickness, coating durability). In order to examine surface hemocompatibility, the materials were contacted with whole human blood under arterial flow simulated conditions followed by calculation of platelet consumption and the number of platelet aggregates. Samples were also contacted with platelet-poor plasma; the number of surface-adsorbed fibrinogen molecules was measured using ELISA assay. Finally, the inflammatory reaction after implantation was assessed, using New Zealand rabbits. The designed coating is characterized by high water content and excellent durability in aqueous environment - over a 35-day period, no significant changes in coating thickness were observed. Experiments with blood proved twice the reduction in adsorption of serum-derived fibrinogen together with a moderate reduction in the number of platelet aggregates formed during the contact of the material with blood. The analysis of an inflammatory reaction after the implantation confirmed high biocompatibility of the fabricated materials - studies have shown no toxic effects of the implanted material on the surrounding animal tissues.