Immobilization of Gelatin onto Poly(Glycidyl Methacrylate)-Grafted Polycaprolactone Substrates for Improved Cell–Material Interactions

Shaojun Yuan 0 1 Gordon Xiong 0 1 Ariel Roguin 0 1 Cleo Choong 0 1 0 A. Roguin Department of Cardiology, Rambam Medical Center, B. Rappaport Faculty of Medicine , Technion, Israel Institute of Technology , 31096 Haifa, Israel 1 S. Yuan G. Xiong C. Choong (&) School of Materials Science and Engineering, Nanyang Technological University , 50 Nanyang Avenue, Singapore 639798, Singapore To enhance the cytocompatibility of polycaprolactone (PCL), cell-adhesive gelatin is covalently immobilized onto the PCL film surface via two surfacemodified approaches: a conventional chemical immobilization process and a surface-initiated atom transfer radical polymerization (ATRP) process. Kinetics studies reveal that the polymer chain growth from the PCL film using the ATRP process is formed in a controlled manner, and that the amount of immobilized gelatin increases with an increasing concentration of epoxide groups on the grafted P(GMA) brushes. In vitro cell adhesion and proliferation studies demonstrate that cell affinity and growth are significantly improved by the immobilization of gelatin on PCL film surfaces, and that this improvement is positively correlated to the amount of covalently immobilized gelatin. With the versatility of the ATRP process and tunable grafting efficacy of gelatin, this study offers a suitable methodology for the functionalization of biodegradable polyesters scaffolds to improve cell-material interactions. 1 Introduction Due to its slow degradation rate in vivo, good processability, and appropriate mechanical properties, polycaprolactone (PCL) is currently being extensively investigated as a scaffold material for tissue engineering applications [18]. However, the intrinsic hydrophobicity of PCL substrates results in poor cell attachment properties, thereby restricting their applications as biomaterials [817]. Modification of PCL substrate surfaces with physiological or biological activities has proven to be an effective strategy to promote cell adhesion and growth. Various methods, such as hydrolysis [13, 18, 19], aminolysis [5, 10, 2023], plasma treatment [8, 1116, 24, 25], UV-induced copolymerization [9, 26], ion-beam irradiation [27], and ozone treatment [28], have been employed in immobilizing extracellular matrix (ECM) molecules (e.g., collagen, gelatin and chitosan) and small active peptide sequences (e.g. Arg-Gly-Asp (RGD)) onto the PCL substrates to induce cell-specific interactions. Alternatively, functional polymer brushes containing reactive hydroxyl, carboxyl or amine groups have been grafted onto the PCL surfaces using c-ray irradiated, ozone or photo-induced grafting to introduce hydrophilicity [14, 2931]. These flexible reactive groups on the polymer brushes are well-suited to conjugate bioactive macromolecules for improved cytocompatibility. However, c-ray irradiated, ozone or photoinduced polymerization grafting of polymer brushes has several limitations, including low density of grafting due to steric hindrance, uncontrollable graft yield of polymer brushes, and undesired formation of a covalent bond between reactive groups on the polymer brushes and the surface [32]. Hence, alternative methods that allow control over brush density, polydispersity and composition are desired. One such alternative is the use of surface-initiated atom transfer radical polymerization (ATRP) method to covalently attach polymer brushes in a tunable and controllable manner [3335]. This approach allows the preparation of polymer brushes bearing reactive pendant groups, such as hydroxyl, carboxylic acid, or epoxide groups, which provide highly reactive binding sites for bioactive macromolecules at the brush interfaces [36]. Hence, surface-initiated ATRP is a promising approach for the functionalization of the PCL surface, as it allows for the control of the length and density of the polymer brushes, which leads to tunable grafting efficiency for the desired biologically active molecules. However, to the best of our knowledge, few studies have been devoted to modifying biodegradable polyester polymers using surface-initiated ATRP for the improvement of their cytocompatibility [37]. Moreover, no systematic study has been performed to investigate the relationship between the surface density of the grafted bioactive molecules and cellular functions in order to demonstrate the tunable grafting efficiency of this approach. In the current study, gelatin was used as the model protein, as its cell-binding properties have been previously demonstrated [38]. Using both a conventional chemical immobilization process and surface-initiated ATRP, gelatin was covalently immobilized onto the PCL substrates with different surface-grafting densities. As schematically illustrated in Scheme 1, the aminolysis reaction with 1,6-hexanediamine resulted in an activated PCL surface with free amino and hydroxyl groups [10, 20, 28]. A monolayer of gelatin was covalently conjugated to the aminolyzed PCL film surface with glutaraldehyde (GA) as bifunctional cross-linking agent, using the conventional chemical immobilization process in Scheme a. To further increase the binding capacity of gelatin on the PCL substrates, well-defined polymer brushes of glycidyl methacrylate (GMA) were prepared via surface-initiated ATRP from the alkyl halide initiator-immobilized PCL film surface in Scheme b. Gelatin was subsequently coupled to the pendant active epoxide groups of the grafted P(GMA) brushes. The efficacy of each functionalization step was ascertained by ATR-FTIR, XPS, AFM, and static water contact angle measurements. Subsequently, in vitro cell adhesion, spreading and proliferation studies were carried out to evaluate the cytocompatibility of the modified PCL surface. 2 Experimental 2.1 Materials Polycaprolactone pellets (PCL, average Mn 45,000), 1,6-hexanediamine (98 %), glycidyl methacrylate (GMA, Scheme 1 Schematic diagram illustrating the aminolysis process used to introduce free amino groups to PCL (i.e. PCL-NH2 surface) and the two subsequent surface-modification schemes used to create gelatinized substrates. In scheme a, gelatin is covalently immobilized onto the aminolyzed PCL surface with glutaradehyde as a crosslinking agent, resulting in the PCL-gelatin surface. In Scheme b, immobilization of an alkyl bromine-containing initiator via condensation reaction leads to the formation of the PCL-Br surface. Surfaceinitiated ATRP of GMA from the PCL-Br surface results in the formation of the PCL-g-P(GMA) surface, and subsequently covalent conjugation of gelatin will result in the PCL-g-P(GMA)-c-gelatin surface [97 %), 2-bromoisobutyrl bromide (BIBB, 98 %), 2,20-bipyridine (Bpy, 98 %), dichloromethane (anhydrous, [99.8 %), triethylamine (TEA, 98 %), isopropyl alcohol, hexane (anhydrous, [95 %), glutaraldehyde (25 %, Grade I), ninhydrin ([95 %), copper (I) bromide (CuBr, 99 %), copper (II) bromide (CuBr2, 98 %), and gelatin (Porcine skin, Type A) were obtained from Sigma-Aldrich Chemic (...truncated)