Carbonyl Iron Foam Surfaces Modified with Poly (L-Lysine) As Smart Surface for Bone Implant

This article presents the surface modification of iron Fe (110) surfaces with Poly-L-Lysine (PLL) with the aim of preparing carbonyl iron bone implants which are less corrosive and more compatible with fibroblast cells. The cytocompatibility of modified surfaces with commercially available α-PLL and electrodeposited ε-PLL was compared by combination of DFT computational simulations with experimental electrochemical and cell adhesion studies to obtain “smart” surface application. Experimental study of fibroblasts adhesion showed better viability of cells on ε-PLL than on α-PLL after modification of Fe surfaces as “smart” surfaces to obtain a different hydrophobicity. The porosity of Fe (110) prevented direct measurements of contact angle and therefore surface hydrophobicity was simulated with calculation of adsorption energies for Fe with both α-/ε-PLL structures. This technique was also employed to calculate the interaction of O-H bonds at the surface. The corrosion potential of Fe (110) with superficially modified ε-PLL was shifted by 0.088V compared to the bare iron surface, thus indicating a stronger resistance to corrosion. The results suggest that modification of Fe surface with ε-PLL has a more pronounced effect on cellular growth on this implant and that the slightly hydrophobic character of ε-PLL leads to better cell adhesion ability.


Introduction
Poly-L-Lysine (PLL) represents a biocompatible polymer which as cationic structure becomes an extraordinarily effective molecular coating for the adhesion of negatively charged biomolecules, generally DNAs [1]. Biosynthesized (ε-PLL) carries R-amino groups on the side chains which can react with the residual amino and carboxyl groups resulting in formation of an anion-cationic pair called the zwitterion. The so formed naturally occuring zwitterion displays antifouling activity when molecules of water interact with the surface of the material, which may suppress the adhesion of the bacteria [2]. As a result of such polycationic nature associated with biodegradability and chirality the homopolymer of the amino acid PLL-based nanomaterials are also being studied to increase biocompatibility, promote cell adhesion, and enhance drug distribution [4] and linear and dendritic PLL structures have been employed, for example, in tissue engineering, or as prophylactic for viral infections [1] Electrostatic self-assembly nanotechnology constitutes a promising way of generating thin layers with highly controlled molecular architectures [5,6]. Another interesting possibility of application of PLL is adsorption on different materials for surface modification and as a method of layer-by-layer fabrication [7]. In general, the appropriate functionalization of metal surfaces with biomolecules that keep their conformation and activity remains a challenge [8]. In the case of iron bone implant surface modification with PLL a corrosion plays an important role.
Application of corrosion inhibitors for the protection of steel is also used and investigated by many authors [9]. The highest protection against metal corrosion is provided by organic molecules that contain heteroatoms such as oxygen, sulfide, and nitrogen [10].
Researchers show that most organic inhibitors work by a mechanism of adsorption on the metal surface. This mechanism involves blocking active sites on the iron surface due to the adsorption of inhibitor molecules. Since the metal surface is covered primarily by adsorbed Cl-ions, amino acid cations are able to electrostatically adsorb on the surface [11]. The lysine adsorbs onto the metallic surfaces by interaction with the ε-amino group [2]. The conformational state of PLL at the molecular level is determined by the degree of electrostatic repulsion between its side chains and hydrogen bonds. The presence of PLL provides superior hydrophilicity and roughness against titanium disc implant surface and safely promotes osteoblast calcium deposition [12]. Surface hydrophilicity plays a significant role in the interaction of cells with the surface. If the surface is only slightly hydrophilic, it is difficult for the cells to adhere. Adhesion and cell spreading cohere with the dispersion and polar components of the energy of the surface [13].
Highly tuned polymer brushes can be used to overcome any limitation with robust coatings used to adjust a great diverse of biological substrates. To enhance integration of implant-bone, titanium substrate can be coated with a SAM-oligoethylene glycol brushes-synthetized by surface-initiated atom transfer radical polymerization that can effectively promote bone cell differentiation in vitro and implant integration [14]. Computational modelling with the Quantum Espresso code [15] (based on plane waves, and with Projector Augmented-Wave (PAW) pseudopotentials generated in the GGA approximation for the exchange-correlation functional), is used to apprehend the adsorbate-surface interactions. Atomic and molecular adsorption on Fe (110) is well documented [15,16]. Since the measurement of contact angle and wettability can be difficult and sensitive to ambient conditions it is important to include computational modelling. In addition to the experimental research, the theoretical study of the hydrophobicity of CeO 2 surfaces is necessary for a better understanding of surface behavior. Fronzi et al. studied the hydrophobicity of Cerium (IV) oxide by DFT method [16].
They calculated water double layer adsorption energies at CeO 2 (100), (110) and (111) surfaces, contact angle and surface hydrophobicity with use of computational modeling. Nowadays, the computational research is also focused on the calculation of the hydrophobicity at the surface of proteins [17], for instance the adsorption of chiral L-lysine on Cu (110) surfaces [18] and L-lysine general adsorption geometry [19]. Computational modelling has provided an insight into the adsorbate-surface interactions at the molecular level. In the present study, we have examined modification of iron foam material prepared by the sintering of carbonyl iron powder with different forms of PLL for adhesion of fibroblast cells. Additionally, the adsorption process of the PLL from the acidic solution onto the iron surface was provided by DFT calculations, in which adsorption geometries of PLL on the Fe (110) slab were considered. Our computational results indicate that PLL confers a higher resistance to corrosion and insights in how the morphology of the PLL layer and its hydrophobicity plays a role in the cytocompatibility of the "smart" surface with potential application as bone implant.

Results of DFT studies of ε-PLL
Firstly, the water adsorption on the iron surface (110) was modeled with initial proposals for the water coating layer displayed in Figure 1 and the α-/ε-PLL layer presented on Figure 2. Then, DFT simulations were investigated to model the adsorption of the α-/ε-PLL on the Fe (110) surface to find how the molecules bind onto the iron surface and to study the surface hydrophobicity. The geometry of α-/ε-poly-L-lysine is displayed on Figure 2. The differences in structure and geometry can be appreciated from their respective side views. Adsorption energies calculated are given in Table 1.
The lowest adsorption energy was found for α-PLL and the contact angle indicates that α-PLL is a hydrophilic surface. Oppositely, the value of adsorption energy of ε-PLL was calculated as high as -0.39eV. Furthermore, the calculated contact angle indicated that this surface was slightly hydrophobic. These results were obtained in the good relations with calculations done in reference [18].   It is worth mentioning that the porosity of the material made very challenging the use of experimental methods to determine the contact angle, and therefore the DFT method here presented has proven to be effective to characterize either surface hydrophobicity or hydrophilicity. Typically, nearly every animal cell favors moderately hydrophilic surfaces in terms of adhesion and growth.
The hydrophobicity over the molecular surface of α-PLL is caused by the surface which is linearly bound through oxygen atoms as and y (C and G) direction. Figure D represents α-PLL and Figure H represents ε-PLL on Fe (110) surface. Atoms: oxygen-red; nitrogen-slightly blue; carbon-brown; hydrogen-white; gold-Fe (110). Table   1 Calculated adsorption energies of different PLL bond on Fe (110).   with such hydrophilic nature [20].    When compared to the previous cases results, ε-PLL ( Figure 5), Figure 6 showed the increase of fibroblast cell viability with cell cultivation duration ( Figure 6) compared to previous cases. The presented electrochemically polymerized ε-PLL coating can be considered as biocompatible and non-cytotoxic. One of the possible reasons of higher cell viability is fact that Figure 5 shows evaluation of cell viability of α-PLL modified iron cellular material and Figure 6 shows iron cellular material modified by ε-PLL, which is considered as nontoxic [23], when compared to its α form [24]. The relation between wettability and cell adhesion has been proven in many studies. Viability on to the α-PLL in case of both concentrations stabilized at intensity of 28000.
Cell viability of the electrochemically polymerized ε-PLL iron cellular material stabilized at significantly higher value of about 35000. Polyethylene surfaces with wettability gradient from 96° to 43° were created using corona discharges [18]. There was also investigated interaction with various cell lines and fetal bovine serum [17].

Cellular material preparation
The iron foam was prepared from iron powder as reported in the literature [26].

Electrochemical preparation of ε-poly-L-lysine layer
Prior to modification, iron foams were sonicated in ethanol for 10 minutes. The electro polymerization of ε-poly-L-lysine was performed in a 1X PBS pH 9.0 containing 10 mM L-lysine by cyclic voltammetry from -0.6 to 2.2V for 5 cycles with a scan rate of 100mV s-1 using potentiostat Autolab 302N (Metrohm, Netherlands).
Electro polymerization was performed in 50ml of solution. After electro polymerization samples were rinsed in purified water and dried in an oven at 50°C for 1 hour. Electrochemically induced polymerization reaction of α-L-lysine, as it is presented in this study, leads to favorization of only ε form of PLL which is biologically active and non-toxic. Very similar way of PLL preparation was presented in [27], where final ε form is present and PLL structure was confirmed by Reflection-absorption infrared spectroscopy.
There also exists another way to prepare ε form PLL, but they are much more difficult [28].

Corrosion measurements of ε-PLL modified carbonyl metallic foam
Potentiodynamic polarization tests were performed using Ag/ AgCl/KCl (3mol L-1) as a reference electrode, a platinum plate as a counter electrode and the iron cellular structure, pure and PLL

General DFT Quantum ESPRESSO calculations of geometries
The molecular models of PLL were well documented [29].

Conclusion
We present a study of both experimental and computational The interaction of different fibroblast cells with the smart surfaces was demonstrated. In future work, we will study smart surfaces for different attraction of fibroblast at bone implant. Most interesting in the next research work will be studying the interaction with PLL dendrigrafts.