title: Biomimetic modified clinical-grade POSS-PCU nanocomposite polymer for bypass graft applications: a preliminary assessment of endothelial cell adhesion and haemocompatibility.
abstract: BACKGROUND
  To date, there are no small internal diameter (<5mm) vascular grafts that are FDA approved for clinical use due to high failure rates from thrombosis and unwanted cell proliferation. The ideal conditions to enhance bioengineered grafts would be the blood contacting lumen of the bypass graft fully covered by endothelial cells (ECs). As a strategy towards this aim, we hypothesized that by immobilising biomolecules on the surface of the polyhedral oligomeric silsesquioxane-poly(carbonate-urea)urethane (POSS-PCU) nanocomposite polymers, which contain binding sites and ligands for cell surface receptors similar to extracellular matrix (ECM) will positively influence the attachment and proliferation of ECs. Since, the surface of POSS-PCU is inert and not directly suitable for immobilisation of biomolecules, plasma graft polymerisation is a suitable method to modify the surface properties ready for immobilisation and biofunctionalisation.
  
  
  METHODS
  POSS-PCU was activated by plasma treatment in air/O2 to from hydroperoxides (-OH, -OOH), and then carboxylated via plasma polymerisation of a 30% acrylic acid solution (Poly-AA) using a two-step plasma treatment (TSPT) process. Collagen type I, a major component of ECM, was covalently immobilised to mimic the ECM structures to ECs (5mg/ml) using a two-step chemical reaction using EDC chemistry. Successful immobilisation of poly-AA and collagen on to the nanocomposites was confirmed using Toluidine Blue staining and the Bradford assay. Un-treated POSS-PCU served as a simple control. The impact of collagen grafting on the physical, mechanical and biological properties of POSS-PCU was evaluated via contact angle (θ) measurements, scanning electron microscopy (SEM), atomic force microscopy (AFM), dynamic mechanical thermal analysis (DMTA), ECs adhesion and proliferation followed by platelet adhesion and haemolysis ratio (HR) tests.
  
  
  RESULTS
  Poly-AA content on each of the plasma treated nanocomposite films increased on Low, Med and High samples due to more carboxylic acid (-COOH) groups at the surface forming amide (-NH2) bonds. The amount of -COOH groups on each of the Low, Med and High nanocomposites correlated with Poly-AA grafting density at 14.7±0.9, 18.9±0.9, and 34.2±2.4 μg/cm(2). Immobilisation of collagen type I on to nanocomposite surface was also found to increase significantly on the Low, Med and High samples from 22±4, 150±15, and 219±17 μg/cm(2), respectively. The level of ECs and their adhesion efficiency were improved with increasing amounts of grafted collagen I. The maximum adhesion of ECs was found on the highest collagen type I coated nanocomposites. Platelet adhesion and activation also increased with increasing collagen density. The obtained HR values for all of the treated samples were well within the acceptable standards for biomaterials (<5% HR).
  
  
  CONCLUSIONS
  Poly-AA-g-POSS-PCU surfaces offer binding sites for the covalent bonding of collagen type I and other biomolecules such as fibronectin by exposure of RGD cell binding domains and growth factors using EDC cross-linking chemistry. Collagen type I modification can yield accelerated EC growth and enhance the endothelialisation of POSS-PCU nanocomposites, and the amount of immobilised collagen can control the level of platelet adhesion on functionalized POSS-PCU via TSPT and poly acrylic acid (poly-AA) treatment. Such surface modification procedures of polymeric surfaces can improve the patency rate of POSS-PCU nanocomposites as vascular bypass grafts in the preparation of a range of medical devices ready for pre-clinical and in vivo evaluation.
authors.affiliation: Biomedical Engineering Faculty, Amirkabir University of Technology, Tehran, Iran.
authors.forename: Atefeh
authors.initials: A
authors.lastname: Solouk
authors.affiliation: Centre for Nanotechnology & Regenerative Medicine, Division of Surgery & Interventional Science, University College London, UK. Electronic address: brian.cousins@ucl.ac.uk.
authors.forename: Brian G
authors.initials: BG
authors.lastname: Cousins
authors.affiliation: Biomedical Engineering Faculty, Amirkabir University of Technology, Tehran, Iran.
authors.forename: Fereshteh
authors.initials: F
authors.lastname: Mirahmadi
authors.affiliation: Polymer Engineering Faculty, Amirkabir University of Technology, Tehran, Iran.
authors.forename: Hamid
authors.initials: H
authors.lastname: Mirzadeh
authors.affiliation: Department of Pathology, School of Medicine, Shahed University, Tehran, Iran.
authors.forename: Mohammad Reza Jalali
authors.initials: MR
authors.lastname: Nadoushan
authors.affiliation: National Cell Bank of Iran, Pasteur Institute of Iran, Tehran.
authors.forename: Mohammad Ali
authors.initials: MA
authors.lastname: Shokrgozar
authors.affiliation: Centre for Nanotechnology & Regenerative Medicine, Division of Surgery & Interventional Science, University College London, UK.
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Seifalian
mesh_terms: D001672:Biocompatible Materials; D032701:Biomimetics; D002448:Cell Adhesion; D001026:Coronary Artery Bypass / Q000295:instrumentation*; D004730:Endothelium, Vascular / Q000166:cytology*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008422:Materials Testing; D018625:Microscopy, Atomic Force; D053761:Nanocomposites; D011108:Polymers / Q000737:chemistry*
chemical_list: D001672:Biocompatible Materials; D011108:Polymers
keywords: Biomimetic modification; Blood compatibility; Bypass graft; Collagen; POSS-PCU