Grafting of phosphorylcholine functional groups on polycarbonate urethane surface for resisting platelet adhesion

In order to improve the resistance of platelet adhesion on material surface, 2-methacryloyloxyethyl phosphorylcholine (MPC) was grafted onto polycarbonate urethane (PCU) surface via Michael reaction to create biomimetic structure. After introducing primary amine groups via coupling tris(2-aminoethyl)amine (TAEA) onto the polymer surface, the double bond of MPC reacted with the amino group to obtain MPC modified PCU. The modified surface was characterized by Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The results verified that MPC was grafted onto PCU surface by Michael reaction method. The MPC grafted PCU surface had a low water contact angle and a high water uptake. This means that the hydrophilic PC functional groups improved the surface hydrophilicity significantly. In addition, surface morphology of MPC grafted PCU film was imaged by atomic force microscope (AFM). The results showed that the grafted surface was rougher than the blank PCU surface. In addition, platelet adhesion study was evaluated by scanning electron microscopy (SEM) observation. The PCU films after treated with platelet-rich plasma demonstrated that much fewer platelets adhered to the MPC-grafted PCU surface than to the blank PCU surface. The antithrombogenicity of the MPC-grafted PCU surface was determined by the activated partial thromboplastin time (APTT). The result suggested that the MPC modified PCU may have potential application as biomaterials in blood-contacting and some subcutaneously implanted devices.     

Plasma-modified and polyethylene glycol-grafted polymers for potential tissue engineering applications

Modified and grafted polymers may serve as building blocks for creating artificial bioinspired nanostructured surfaces for tissue engineering. Polyethylene (PE) and polystyrene (PS) were modified by Ar plasma and the surface of the plasma activated polymers was grafted with polyethylene glycol (PEG). The changes in the surface wettability (contact angle) of the modified polymers were examined by goniometry. Atomic Force Microscopy (AFM) was used to determine the surface roughness and morphology and electrokinetical analysis (Zeta potential) characterized surface chemistry of the modified polymers. Plasma treatment and subsequent PEG grafting lead to dramatic changes in the polymer surface morphology, roughness and wettability. The plasma treated and PEG grafted polymers were seeded with rat vascular smooth muscle cells (VSMCs) and their adhesion and proliferation were studied. Biological tests, performed in vitro, show increased adhesion and proliferation of cells on modified polymers. Grafting with PEG increases cell proliferation, especially on PS. The cell proliferation was shown to be an increasing function of PEG molecular weight.     

Modulation of Lumen Formation by Microgeometrical Bioactive Cues and Migration Mode of Actin Machinery

How endothelial cells (ECs) express the particular filopodial or lamellipodial form of the actin machinery is critical to understanding EC functions such as angiogenesis and sprouting. It is not known how these mechanisms coordinately promote lumen formation of ECs. Here, adhesion molecules (RGD peptides) and inductor molecules (BMP‐2 mimetic peptides) are micropatterned onto polymer surfaces by a photolithographic technique to induce filopodial and lamellipodial migration modes. Firstly, the effects of peptide microgeometrical distribution on EC adhesion, orientation and morphogenesis are evaluated. Large micropatterns (100 μm) promote EC orientation without lumen formation, whereas small micropatterns (10–50 μm) elicit a collective cell organization and induce EC lumen formation, in the case of RGD peptides. Secondly, the correlation between EC actin machinery expression and EC self‐assembly into lumen formation is addressed. Only the filopodial migration mode (mimicked by RGD) but not lamellipodial migration mode (mimicked by BMP‐2) promotes EC lumen formation. This work gives a new concept for the design of biomaterials for tissue engineering and may provide new insight for angiogenesis inhibition on tumors.     

Arg-Gly-Asp (RGD) Modified Biomimetic Polymeric Materials

The new generation of biomaterials focuses on the design of biomimetic polymeric materials that are capable of eliciting specific cellular responses and directing new tissue formation. Since Arg-Gly-Asp (RGD) sequences have been found to promote cell adhesion in 1984, numerous polymers have been functionalized with RGD peptides for tissue engineering applications. This review gave the advance in RGD modified biomimetic polymeric materials, focusing on the mechanism of RGD, the surface and bulk modification of polymer with RGD peptides and the evaluation in vitro and in vivo of the modified biomimetic materials.     

Quantitative grafting of peptide onto the nontoxic biodegradable waterborne polyurethanes to fabricate peptide modified scaffold for soft tissue engineering

Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide has frequently been used in the biomedical materials to enhance adhesion and proliferation of cells. In this work, we modified the nontoxic biodegradable waterborne polyurethanes (WBPU) with GRGDSP peptide and fabricated 3-D porous scaffold with the modified WBPU to investigate the effect of the immobilized GRGDSP peptide on human umbilical vein endothelial cells (HUVECs) adhesion and proliferation. A facile and reliable approach was first developed to quantitative grafting of GRGDSP onto the WBPU molecular backbone using ethylene glycol diglycidyl ether (EX810) as a connector. Then 3-D porous WBPU scaffolds with various GRGDSP content were fabricated by freeze-drying the emulsion. In both of the HUVECs adhesion and proliferation tests, enhanced cell performance was observed on the GRGDSP grafted scaffolds compared with the unmodified scaffolds and the tissue culture plate (TCP). The adhesion rate and proliferation rate increased with the increase of GRGDSP content in the scaffold and reached a maximum with peptide concentration of 0.85 μmol/g based on the weight of the polyurethanes. These results illustrate the necessity of the effective control of the GRGDSP content in the modified WBPU and support the potential utility of these 3-D porous modified WBPU scaffolds in the soft tissue engineering to guide cell adhesion, proliferation and tissue regeneration.     

Hemocompatible surface of electrospun nanofibrous scaffolds by ATRP modification

The electrospun scaffolds are potential application in vascular tissue engineering since they can mimic the nano-sized dimension of natural extracellular matrix (ECM). We prepared a fibrous scaffold from polycarbonateurethane (PCU) by electrospinning technology. In order to improve the hydrophilicity and hemocompatibility of the fibrous scaffold, poly(ethylene glycol) methacrylate (PEGMA) was grafted onto the fiber surface by surface-initiated atom transfer radical polymerization (SI-ATRP) method. Although SI-ATRP has been developed and used for surface modification for many years, there are only few studies about the modification of electrospun fiber by this method. The modified fibrous scaffolds were characterized by SEM, Fourier transform infrared (FTIR), and X-ray photoelectron spectroscopy (XPS). The scaffold morphology showed no significant difference when PEGMA was grafted onto the scaffold surface. Based on the water contact angle measurement, the surface hydrophilicity of the scaffold surface was improved significantly after grafting hydrophilic PEGMA (P = 0.0012). The modified surface showed effective resistance for platelet adhesion compared with the unmodified surface. Activated partial thromboplastin time (APTT) of the PCU-g-PEGMA scaffold was much longer than that of the unmodified PCU scaffold. The cyto-compatibility of electrospun nanofibrous scaffolds was tested by human umbilical vein endothelial cells (HUVECs). The images of 7-day cultured cells on the scaffold surface were observed by SEM. The modified scaffolds showed high tendency to induce cell adhesion. Moreover, the cells reached out pseudopodia along the fibrous direction and formed a continuous monolayer. Hemolysis test showed that the grafted chains of PEGMA reduced blood coagulation. These results indicated that the modified electrospun nanofibrous scaffolds were potential application as artificial blood vessels.     

A novel polyurethane modified with biomacromolecules for small-diameter vascular graft applications

There is a growing demand for small-caliber tissue-engineered vascular grafts to replace damaged vessels. Fabricated scaffolds are unable to precisely mimic the mechanical properties of native vessels, provide long-term patency and support cell adhesion and growth, in particular support endothelialization. In this study, a new biodegradable poly(ether ester urethane) urea (PEEUU) was synthesized. The synthesized polyurethane was then functionalized by introducing free amino groups through aminolysis for further surface modification by immobilization of biomacromolecules on the surface of vascular grafts. The modified surfaces were then characterized using attenuated total reflectance-Fourier transform infrared spectroscopy, water contact angle measurement and atomic force microscopy. The mechanical properties of the fabricated scaffolds were analyzed, revealing mechanical properties close to that of the natural vessels. Surface modifications led to improved cell–scaffold interactions, showing appropriate cell attachment and function on the scaffolds. A confluent layer of endothelial cells was formed on biomacromolecule-immobilized PEEUU vascular grafts. The preliminary results of this study demonstrated that the new polyurethane modified with biomacromolecules can be considered as a candidate material for vascular tissue engineering application with capability to support endothelialization of fabricated vascular grafts.     

Grafting of poly(ethylene glycol) monoacrylates on polycarbonateurethane by UV initiated polymerization for improving hemocompatibility

Poly(ethylene glycol) monoacrylates (PEGMAs) with a molecular weight between 400 and 1,000 g mol^?1 were grafted by ultraviolet initiated photopolymerization on the surface of polycarbonateurethane (PCU) for increasing its hydrophilicity and improving its hemocompatibility. The surface-grafted PCU films were characterized by Fourier transformation infrared spectroscopy, X-ray photoelectron spectroscopy, water contact angle, scanning electron microscopy (SEM) and atomic force microscopy measurements. The surface properties of the modified films were studied in dry and wetted state. Blood compatibility of the surfaces was evaluated by platelet adhesion tests and adhered platelets were determined by SEM. The results showed that the hydrophilicity of the films had been increased significantly by grafting PEGMAs, and platelets adhesion onto the film surface was obviously suppressed. Furthermore, the molecular weight of PEGMAs had a great effect on the hydrophilicity and hemocompatibility of the PCU films after surface modification and increased with increasing molecular weight of PEGMAs.     

Functionalization of the surface of electrospun poly(epsilon-caprolactone) mats using zwitterionic poly(carboxybetaine methacrylate) and cell-specific peptide for endothelial progenitor cells capture

A novel approach for vascular grafts to achieve rapid endothelialization is to attract endothelial progenitor cells (EPCs) from peripheral blood onto grafts via EPC-specific antibodies, aptamer, or peptides that specifically bind to EPCs. Inspired by this idea, the electrospun poly(epsilon-caprolactone) (PCL) mats were modified with zwitterionic poly(carboxybetaine methacrylate) (PCBMA) and phage display-selected-EPC-specific peptide, TPSLEQRTVYAK (TPS). We tested the physical and chemical properties, cyto-compatibility, and platelet adhesion of the modified material, and investigated the specificity of the functionalized surface for capturing EPCs. The results indicated that PCL modified with zwitterionic PCBMA and TPS peptide showed improved hydrophilicity without morphology change and damage of the mats. Furthermore, the modified material supported adherence and growth of vascular cells and resisted platelets adhesion. The surfaces also specifically captured EPCs rather than bone marrow mesenchymal stem cells and human umbilical vein endothelial cells. This surface-functionalized PCL graft may offer new opportunities for designing new vascular grafts.     

Peptide immobilization on polyethylene terephthalate surfaces to study specific endothelial cell adhesion, spreading and migration

To control specific endothelial cell (EC) functions, cell adhesive RGDS, EC specific REDV and YIGSR peptides, and angiogenic SVVYGLR sequences were covalently immobilized onto polyethylene terephthalate (PET) surfaces for the purpose of cell culture. X-ray photoelectron spectroscopy, atomic force microscopy, fluorescence microscopy and contact angle measurement were employed for characterization of surface modifications. The peptide density on PET surfaces was evaluated by fluorescence microscopy. The surfaces immobilized with peptides were exposed to human umbilical vein endothelial cells to study their specific effects onto EC functions. The results showed that the surface functionalized by these peptides enhanced the EC adhesion, spreading and migration as compared with native PET surfaces. Specifically, the RGDS peptides induced more cell adhesion than other peptides. The YIGSR and SVVYGLR sequences induced more cell spreading and cell migration, represented by intense focal adhesion at the leading edges of cell spreading and migration. The bi-functionalization of RGDS and SVVYGLR peptides (MIX) combined the advantages of both peptides and induced significant EC adhesion, spreading and migration. Our study indicates that the surface functionalization by peptides specific for ECs, especially the combination of RGDS with SVVYGLR or YIGSR peptides, has potential applications in promoting endothelialization of vascular prostheses and for construction of vascularized tissues in tissue engineering.     

A Dynamically Tunable,Bioinspired Micropatterned Surface Regulates Vascular Endothelial and Smooth Muscle Cells Growth at Vascularization

Regulation of the growth of vascular endothelial cells (ECs) and smooth muscle cells (SMCs) with artificial vascular grafts at vascularization is well‐known to regenerate functional blood vessels for treating cardiovascular disease; however, little research has been published on this subject. Here, a novel polymer vascular graft is presented, whose inner surface contains an assembled circular microgroove pattern decorated with a combination of concentric circular microgrooves and radial, straight microgrooves inspired by the orientation of SMCs and ECs in natural tissues. The surface micropatterns can produce dynamically tunable variations via the thermally switched shape memory. The results from the in vitro EC/SMC co‐cultures reveal that the surface micropatterns have a great capacity to regulate the specific distribution of ECs/SMCs because the ECs grow along the radial, straight microgrooves and the SMCs grow along concentric circular microgrooves. The in vivo vascularization is further analyzed by implanting the vascular graft in the rabbit carotid artery. Both histological analysis and immunofluorescence staining demonstrate that it is capable of highly effectively capturing ECs and SMCs in the blood and subsequent regeneration of new blood vessels. Therefore, this study opens a new possibility for regenerating neovessels to replace and repair damaged vessels for cardiovascular diseases treatment.     

Temperature-Responsive Chromatography Using Poly(N-isopropylacrylamide)-Modified Silica

A new concept in chromatography is proposed that utilizes a temperature-responsive surface with a constant aqueous mobile phase. The surface of the silica stationary phase in high-performance liquid chromatography (HPLC) has been modified with temperature-responsive polymers to exhibit temperature-controlled hydrophilic/hydrophobic changes. Poly(N-isopropylacrylamide) (PIPAAm) was grafted onto (aminopropyl)silica using an activated ester-amine coupling method. These grafted silica surfaces show hydrophilic properties at lower temperatures which, as temperature increases, transform to hydrophobic surface properties. The elution profile of five mixed steroids on an HPLC column packed with this material depends largely on the temperature of the aqueous mobile phase. Retention times increase with increasing temperature without any change in the eluent. Changes in the retention times of hydrophobic steroids were larger than those for hydrophilic steroids. The temperature-responsive interaction between PIPAAm-modified silica and these steroids is proposed to result from changes in the surface properties of the HPLC stationary phase by the transition of hydrophilic/hydrophobic surface-grafted IPAAm polymers. We demonstrate a novel and useful new chromatography system in which surface properties and the resulting function of the HPLC stationary phase are controlled by external temperature changes. This method should be effective in biological and biomedical separations of peptides and proteins using only aqueous mobile phases.     

Investigation of effects of Argenine–Glycine–Aspartate (RGD) and nano-scale titanium coatings on cell spreading and adhesion

This paper examines the effects of physical and chemical surface modifications on the biocompatibility of silicon surfaces that are relevant to implantable silicon Bio-micro-electro-mechanical systems (BioMEMS). Two types of surface modifications were explored. The first involved the deposition of nano-scale biocompatible layers of pure titanium on silicon, while the second explored the covalent attachment of the binding peptide Argenine–Glycine–Aspartic acid (RGD) for improved cell adhesion. Improvements in biocompatibility were assessed through examination of cell areas after culture, as well as the measurements of adhesion strengths, as determined by shear assay techniques. The titanium nanolayers and the RGD coating resulted in improvements in biocompatibility. Increased cell spreading areas and improved adhesion strength were obtained from short and long-term studies of Human Osteosarcoma (HOS) cells cultured on the coated surfaces. RGD functionalization resulted in the greatest improvement in cell spreading area and adhesion strength for short culture times. The effects of the titanium, while less than those of RGD for short culture times, appeared to be greater after 48 h of culture.     

RGD修饰钛表面对人牙龈成纤维细胞初期黏附和铺展的影响

用羰基二咪唑(1,1'-carbonyldiimidazole,CDI)将含RGD的短肽共价连接到纯钛表面,研究接枝后的钛表面对原代培养的人牙龈成纤维细胞(human gingival fibroblasts,HGF)初期黏附和铺展的影响.结果表明,RGD修饰的纯钛表面粘附的细胞数比未修饰钛表面多,细胞铺展面积比钛表面的大,应力纤维的形成比钛表面早.RGD接枝钛表面更有利于人牙龈成纤维细胞的粘附,改善了纯钛的生物相容性.     

Dextran‐based coating system for the immobilization of cell adhesion promoting molecules on titanium surfaces

Immobilization of adhesive peptides interacting with cellular integrin receptors onto metallic implant surfaces represents a promising approach to improve osseointegration of implants into the surrounding tissue. In the present study, a functional dextran‐based coating system consisting of an amino titanate adhesion promoter with dendritic structure and a carboxymethyl dextran was established to bind an RGD‐containing adhesive peptide via a selective coupling methodology onto titanium surfaces. The three‐step reaction procedure was characterized by X‐ray photoelectron spectroscopy. In cell adhesion experiments it could be demonstrated that dextran coatings containing immobilized RGD promote attachment and spreading of fibroblast and pre‐osteoblastic cells compared to native as well as CMD‐coated titanium surfaces without RGD. The direct attachment of the RGD sequence to the metal surface via the amino titanate adhesion promoter did not increase pre‐osteoblastic cell spreading, whereas coupling of RGD to the polymeric carboxy­methyl dextran layer slightly enhanced spreading of the cells.     

Protein-mediated macrophage adhesion and activation on biomaterials: a model for modulating cell behavior

The elucidation of proteins involved in biomaterial-modulated macrophage behavior is critical for the improvement of material performance and the initial exploration of material design capable of manipulating macrophage function for tissue engineering. In this paper, several in vitro and in vivo techniques are presented to demonstrate means of delineating a part of the complex molecular mechanisms involved in the interaction between biomaterial and macrophage adhesion and phenotypic development. The following conclusions were reached: (1) using radioimmunoassay, complement component C3 was found to be critical in mediating human macrophage adhesion on polyurethanes. (2) The presence of a diphenolic antioxidant additive in polyurethanes increased the propensity for complement upregulation but did not affect adherent macrophage density. (3) The subcutaneous cage-implant system was utilized to delineate interleukin-4 participation in the fusion of adherent macrophages to form foreign body giant cells in vivo in mice. The injection of purified interleukin-4 neutralizing antibody into the implanted cages significantly decreased the giant cell density; conversely, the giant cell density was significantly increased by the injection of recombinant interleukin-4 when compared with the controls. (4) The RGD and PHSRN amino acid sequences of the central cell binding domain and the PRRARV sequence of the C-terminal heparin binding domain of human plasma fibronectin were utilized to study the structure-functional relationship of protein in mediating macrophage behavior. Polyethyleneglycol-based networks grafted with the RGD-containing peptide supported higher adherent human macrophage density than surfaces grafted with other peptides. The formation of foreign body giant cell was highly dependent on the relative orientation between PHSRN and RGD domains located in a single peptide. © 1999 Kluwer Academic Publishers     

Facile surface modification of silicone rubber with zwitterionic polymers for improving blood compatibility

A facile approach to modify silicone rubber (SR) membrane for improving the blood compatibility was investigated. The hydrophobic SR surface was firstly activated by air plasma, after which an initiator was immobilized on the activated surface for atom transfer radical polymerization (ATRP). Three zwitterionic polymers were then grafted from SR membrane via surface-initiated atom transfer radical polymerization (SI-ATRP). The surface composition, wettability, and morphology of the membranes before and after modification were characterized by X-ray photoelectron spectroscopy (XPS), static water contact angle (WCA) measurement, and atomic force microscopy (AFM). Results showed that zwitterionic polymers were successfully grafted from SR surfaces, which remarkably improved the wettability of the SR surface. The blood compatibility of the membranes was evaluated by protein adsorption and platelet adhesion tests in vitro. As observed, all the zwitterionic polymer modified surfaces have improved resistance to nonspecific protein adsorption and have excellent resistance to platelet adhesion, showing significantly improved blood compatibility. This work should inspire many creative uses of SR based materials for biomedical applications such as vessel, catheter, and microfluidics.     

Effects of adsorbed heat labile serum proteins and fibrinogen on adhesion and apoptosis of monocytes/macrophages on biomaterials

A previously established human monocyte culture protocol was used to determine the effects of varying adsorbed proteins on monocyte/macrophage adhesion and survival on dimethyl-silane (DM) or RGD modified glass coverslips. Cells were allowed to adhere for 2 h in the absence of protein or in the presence of serum, fibrinogen (Fg), heat inactivated serum (HIS), serum supplemented with Fg or HIS with Fg. Cell adhesion and apoptosis rates were determined on days 0 (2 h), 3, 7 and 10 of culture. The presence of serum alone in the initial culture was sufficient to optimize monocyte/macrophage adhesion and survival rates. Adding Fg to serum did not increase adhesion nor decrease apoptotic rates. No protein or the addition of HIS during the initial incubation period significantly decreased monocyte/macrophage adhesion and survival on both surfaces, however, the addition of Fg to HIS restored adhesion and survival rates to those seen with in the presence of serum alone on RGD surfaces. These studies demonstrate that monocyte/macrophage adhesion and survival on biomaterial surfaces are optimized by adsorbed heat labile serum proteins while adsorbed Fg plays a surface property-dependent role.     

Surface modification of poly(dimethylsiloxane) microfluidic devices by ultraviolet polymer grafting

Poly(dimethylsiloxane) (PDMS)-based microfluidic devices are increasing in popularity due to their ease of fabrication and low costs. Despite this, there is a tremendous need for strategies to rapidly and easily tailor the surface properties of these devices. We demonstrate a one-step procedure to covalently link polymers to the surface of PDMS microchannels by ultraviolet graft polymerization. Acrylic acid, acrylamide, dimethylacrylamide, 2-hydroxylethyl acrylate, and poly(ethylene glycol)monomethoxyl acrylate were grafted onto PDMS to yield hydrophilic surfaces. Water droplets possessed contact angles as low as 45 degrees on the grafted surfaces. Microchannels constructed from the grafted PDMS were readily filled with aqueous solutions in contrast to devices composed of native PDMS. The grafted surfaces also displayed a substantially reduced adsorption of two test peptides compared to that of oxidized PDMS. Microchannels with grafted surfaces exhibited electroosmotic mobilities intermediate to those displayed by native and oxidized PDMS. Unlike the electroosmotic mobility of oxidized PDMS, the electroosmotic mobility of the grafted surfaces remained stable upon exposure to air. The electrophoretic resolution of two test peptides in the grafted microchannels was considerably improved compared to that in microchannels composed of oxidized PDMS. By using the appropriate monomer, it should be possible to use UV grafting to impart a variety of surface properties to PDMS microfluidics devices.     

The synergistic effect of aligned nanofibers and hyaluronic acid modification on endothelial cell behavior for vascular tissue engineering

The endothelialization of tissue-engineered vascular grafts (TEVGs) is considered to be an effective strategy to prevent the coagulation and restenosis of small-diameter vascular grafts. In this study, we fabricated well aligned nanofibrous scaffolds with PCL using a high speed rotating collector, modified those surfaces with hyaluronic acid (HA) and studied the synergistic effect of the scaffolds on the endothelial cells behavior in vitro. The well-aligned oriented architecture was observed by SEM images in the nanofibrous scaffolds. The contact angle measurements and FTIR-ATR evidenced that HA was successfully modified on the PCL nanofibrous scaffolds and hydrophilicity of the scaffolds was increased after HA coating. The results of adhesion and morphology of human umbilical vein endothelial cells (HUVECs) showed that the HA-coating aligned PCL (HA-aPCL) nanofibrous scaffolds could highly promote attachment and guide HUVECs bipolar spread with the parallel aligned nanofibers. Furthermore, HUVECs on the HA-aPCL formed a confluent monoendothelial cell layer and exhibited superior protein expression levels of von Willebrand factor (vWF). This study suggested that the combination of aligned nanostructure and HA modification was more capable of promoting the regeneration of functional endothelium for vascular tissue engineering than individual use.