Musammir Khan

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.

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.

Hydrophilic PCU scaffolds prepared by grafting PEGMA and immobilizing gelatin to enhance cell adhesion and proliferation

Gelatin contains many functional motifs which can modulate cell specific adhesion, so we modified polycarbonate urethane (PCU) scaffold surface by immobilization of gelatin. PCU-g-gelatin scaffolds were prepared by direct immobilizing gelatins onto the surface of aminated PCU scaffolds. To increase the immobilization amount of gelatin, poly(ethylene glycol) methacrylate (PEGMA) was grafted onto PCU scaffolds by surface initiated atom transfer radical polymerization. Then, following amination and immobilization, PCU-g-PEGMA-g-gelatin scaffolds were obtained. Both modified scaffolds were characterized by chemical and biological methods. After immobilization of gelatin, the microfiber surface became rough, but the original morphology of scaffolds was maintained successfully. PCU-g-PEGMA-g-gelatin scaffolds were more hydrophilic than PCU-g-gelatin scaffolds. Because hydrophilic PEGMA and gelatin were grafted and immobilized onto the surface, the PCU-g-PEGMA-g-gelatin scaffolds showed low platelet adhesion, perfect anti-hemolytic activity and excellent cell growth and proliferation capacity. It could be envisioned that PCU-g-PEGMA-g-gelatin scaffolds might have potential applications in tissue engineering artificial scaffolds.

Surface tailoring for selective endothelialization and platelet inhibition via a combination of SI-ATRP and click chemistry using Cys–Ala–Gly-peptide

Surface tailoring is an attractive approach to enhancing selective endothelialization, which is a prerequisite for current vascular prosthesis applications. Here, we modified polycarbonate urethane (PCU) surface with both poly(ethylene glycol) and Cys-Ala-Gly-peptide (CAG) for the purpose of creating a hydrophilic surface with targeting adhesion of endothelial cells (ECs). In the first step, PCU-film surface was grafted with poly(ethylene glycol) methacrylate (PEGMA) to covalently tether hydrophilic polymer brushes via surface initiated atom transfer radical polymerization (SI-ATRP), followed by grafting of an active monomer pentafluorophenyl methacrylate (PFMA) by a second ATRP. The postpolymerization modification of the terminal reactive groups with allyl amine molecules created pendant allyl groups, which were subsequently functionalized with cysteine terminated CAG-peptide via photo-initiated thiol-ene click chemistry. The functionalized surfaces were characterized by water contact angle and XPS analysis. The growth and proliferation of human ECs or human umbilical arterial smooth muscle cells on the functionalized surfaces were investigated for 1, 3 and 7 day/s. The results indicated that these peptide functionalized surfaces exhibited enhanced EC adhesion, growth and proliferation. Furthermore, they suppressed platelet adhesion in contact with platelet-rich plasma for 2h. Therefore, these surfaces with EC targeting ligand could be an effective anti-thrombogenic platform for vascular tissue engineering application.

Proliferation and migration of human vascular endothelial cells mediated by ZNF580 gene complexed with mPEG-b-P(MMD-co-GA)-g-PEI microparticles

Herein, we developed a novel biodegradable gene carrier for rapid endothelialization of endothelial cells (ECs) in vitro. Three triblock amphiphilic copolymers, methoxy-poly(ethylene glycol)-block-poly(3(S)-methyl-2,5-morpholinedione-co-glycolide)-graft-polyethyleneimine (mPEG-b-P(MMD-co-GA)-g-PEI) with different 3(S)-methyl-2,5-morpholinedione and glycolide contents were synthesized. Microparticles (MPs) were obtained via self-assembly of these copolymers. The hydrophobic core composed of P(MMD-co-GA) segments provide crosslinking points for numbers of PEG and short PEI chains to form a highly hydrophilic and positively charged corona/shell of MPs. Using these MPs, potential genes (ZNF580) for rapid endothelialization were efficiently transported into EA.hy926 cells. Because of the hydrophilic PEG chains and low molecular weight PEI in the triblock copolymers, the cytotoxicity of these MPs and their complexes with pEGFP-ZNF580 was decreased significantly. The transfection efficacy of MPs/pEGFP-ZNF580 complexes was as high as Lipofectamine™ 2000 reagent to EA.hy926 cells in vitro. The proliferation and migration of EA.hy926 cells were improved greatly by the expression of pEGFP-ZNF580 after 60 hours. Our results indicated that the mPEG-b-P(MMD-co-GA)-g-PEI based MPs could be a suitable non-viral gene carrier for ZNF580 gene to enhance rapid endothelialization.

Fabrication and characterization of electrospun gelatin-heparin nanofibers as vascular tissue engineering

AbstractIn this paper, heparin was introduced into electrospun gelatin nanofibrous scaffold for assessment as a controlled delivery device in vascular tissue engineering application. Hybrid gelatin-heparin fibers with smooth surfaces and no bead defects were produced from gelatin solutions with 18% w/v in acetic acid aqueous solution. A significant decrease in fiber diameter was observed when the heparin content was increased from 1 to 5 wt%. The properties of composite gelatin-heparin scaffolds were confirmed by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) measurement. The gelatin-heparin fibrous scaffolds were also cross-linked using 1 wt% glutaraldehyde vapor-phase for 7 days. A sustained release of heparin could be achieved from gelatinheparin scaffolds over 14 days. The results of the biocompatibility in vitro tests carried out using human umbilical vein endothelial cells indicated good cell viability and proliferation on the gelatin-heparin scaffolds. The results demonstrated that the use of electrospun gelatin fibers as heparin carriers could be promising for vascular tissue applications.

Immobilized Bioactive Agents onto Polyurethane Surface with Heparin and Phosphorylcholine Group

AbstractHeparin (HEP) and phosphorylcholine groups (PC) were grafted onto the polyurethane (PU) surface in order to improve biocompatibility and anticoagulant activity. After the surface grafting sites of PU were amplified with the primary amine groups of polyethylenimine (PEI), heparin was covalently linked onto the surface by the reaction between the amino group and the carboxyl group. PC groups were covalently immobilized on the PU-PEI surface through the reaction between the amino group and the aldehyde group of phosphorylcholine glyceraldehyde (PCGA). The surface density of primary amine groups was determined by a ninhydrin assay. The amino group density reached a maximum of 0.88 μmol/cm2 upon incorporation of 10 wt% PEI. The amount of heparin covalently immobilized on the PU-PEI surface was determined by the toluidine blue method. The grafting chemistry resulted in the comparatively dense immobilization of HEP (2.6 μg/cm2) and PC to the PU-PEI surfaces. The HEP and PC modified surfaces were characterized by water uptake (PU 0.15 mg/cm2, PU-PEI 3.54 mg/cm2, PU-HEP 2.04 mg/cm2, PU-PC 2.38 mg/cm2), water contact angle (PU 95.3°, PU-PEI 34.0°, PU-HEP 39.5°, PU-PC 37.2°), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and scanning electron microscope (SEM). The results demonstrated that the PUPEI surface was successfully grafted with HEP and PC. The hydrophilicity and hemocompatibility of these grafted surfaces were significantly improved. These results suggested that the PU-HEP and PU-PC composite films are promising candidates for blood contacting tissue engineering.

Manipulation of polycarbonate urethane bulk properties via incorporated zwitterionic polynorbornene for tissue engineering applications

Elastomeric crosslinked materials based on polycarbonate urethane (PCU) and zwitterionic polynorbornene were designed by thiol–ene click-chemistry and crosslinking reaction. The zwitterionic polynorbornene poly(NSulfoZI) with functionalisable double bonds was first treated with L-cysteine via thiol–ene click-reaction and subsequently formed a crosslinked structure upon treatment with PCU in the presence of a small amount of hexamethylene-1,6-diisocyanate as a crosslinking agent. The obtained materials possessed improved tensile strength (14–20 MPa) and initial modulus (8–14 MPa). All of these materials showed high breaking strain (eb 740–900%) except the material with a high poly(NSulfoZI) content of 28% (eb 470 ± 80%). The biodegradability of these materials was enhanced compared to blank PCU, as demonstrated by testing in PBS for five weeks. Moreover, the cytocompatibility was studied by MTT assay. The adhesion and proliferation of endothelial cells (EA.hy926) over a one-week period indicated that cell growth on these designed material surfaces was enhanced. Therefore, these zwitterionic polynorbornene-modified PCU-based materials could be suitable candidates for tissue engineering applications.

Antimicrobial surfaces grafted random copolymers with REDV peptide beneficial for endothelialization

Polycarbonate urethane (PCU) elastomeric materials have been developed for vascular prosthesis applications, because of their excellent mechanical and physical properties. However, thrombosis and inflammation often limit their usage as small-diameter vascular grafts. Herein, we focused on the design and functionalization of a PCU elastomer with enhanced hemocompatibility, rapid endothelialization and antimicrobial properties. An atom transfer radical polymerization (ATRP) technique was utilized to graft random copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) and eugenyl methacrylate (EgMA) onto a PCU surface, and subsequently the cysteine-terminated CREDV peptide sequence was directly linked onto the surface by a thiol-ene click reaction to prepare a series of REDV peptide functionalized surfaces. The chemical compositions of the modified surfaces were quantified by X-ray photoelectron spectroscopy (XPS), and the hydrophilicity was evaluated by water contact analysis and water uptake. The surface hemocompatibility was verified by platelet adhesion assays, and the results demonstrated that platelet adhesion was significantly reduced on the modified surface. More importantly, the functionalized surfaces with high hydrophilicity and cell specific adhesive REDV peptide could selectively enhance the adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) but they suppressed these behaviors in human arterial smooth muscle cells (HASMCs). Moreover, these surfaces showed excellent antibacterial properties, which originate from the EgMA moieties of the copolymers. The successful fabrication of multifunctional surfaces with excellent hemocompatibility, rapid endothelialization, and good antimicrobial activity through a feasible route could be an attractive platform for tissue engineering applications.

Biomimetic surface modification of polycarbonateurethane film via phosphorylcholine-graft for resisting platelet adhesion

AbstractPhosphorylcholine groups were covalently introduced onto a polycarbonateurethane (PCU) surface in order to create a biomimetic structure on the polymer surfaces. After introducing primary amine groups onto the polymer surface by 1,6-hexanediamine, phosphorylcholine groups were covalently linked onto the surface by the reductive amination between the amino group and the aldehyde group of phosphorylcholine glyceraldehyde (PCGA). The results of water contact angle test, X-ray photoelectron spectroscopy (XPS), and X-ray fluorescence spectrometer (XRF) analysis of the modified films indicated that PCGA had already been covalently linked to the PCU surface. The topographies and surface roughnesses were both imaged and measured by atomic force microscopy (AFM). Scanning electron microscopy (SEM) observation of the PCU films after treatment with platelet-rich plasma demonstrated that platelets had rarely adhered to the surface of the PCGA-grafted PCU films but had mainly adhered to the surface of the blank PCU films. The platelet adhesion result indicated that the PC modified PCU films could resist platelet adhesion after grafting with PCGA, and that these PCGA-grafted PCU materials, potentially, might be applied as blood-contacting materials.