Development of Polymer/Nanodiamond Composite Coatings to Control Cell Adhesion, Growth, and Functions

Milena Keremidarska*; Kamelia Hristova*; Todor Hikov†; Ekaterina Radeva†; Dimitar Mitev†; Ivailo Tsvetanov†; Radina Presker‡; Damjana Drobne§; Barbara Drašler§; Sara Novak§; Veno Kononenko§; Kristina Eleršič¶; Lilyana Pramatarova†; Natalia Krasteva*,1    * Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Sofia, Bulgaria
† Institute of Solid State Physics, Bulgarian Academy of Sciences, Sofia, Bulgaria
‡ Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia
§ Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
¶ Institute J. Stefan, Ljubljana, Slovenia
1 Corresponding author: email address: natalia.krasteva@yahoo.com

1 Introduction

Bone tissue-engineered constructs have a great potential for the treatment of large bone defects caused by tumor, injury, or posttraumatic osteomyelitis [1,2]. In such constructs, the key elements are biomaterials that have to provide an appropriate microenvironment for tissue cells to attach, grow, and differentiate [3,4]. It is also crucial the biomaterial to have mechanical properties similar to the native bone [5]. Despite the remarkable progress in recent years, the engineering of materials matching both the mechanical and biological properties comparable with those of natural, healthy bone still remains a challenge.

Composite materials are of interest because they combine the advantages of different materials to achieve specific structural properties, while a single type of material usually cannot provide all necessary properties [6]. From the biological point of view, it makes sense to combine polymer and inorganic compounds to fabricate biomaterials for bone tissue engineering, because human bone tissue is a biologically and chemically bonded composite of inorganic hydroxyapatite embedded in an organic matrix of collagen and noncollagenous proteins [7,8]. The first step toward the development of composites for bone substitution is the identification of the relevant class of biomaterials. A variety of natural and synthetic polymers are now available for bone tissue engineering applications but all of them have some deficiencies [9,10]. Synthetic polymers have gained a significant advantage over naturally occurring polymers because they can be produced under controlled conditions and therefore their properties are in general predictable and reproducible [11,12]. One class of synthetic polymers widely used in biomedical applications is organosilicones due to their excellent inertness, flexibility, smoothness, and thermal and oxidative stability [13]. Organosilicones have been used for the production of oxygen masks, teats for baby bottles, tubes for extracorporeal circulation in heart surgery and dialysis, drains and catheters, prosthesis, contact lens, insulation coating for leads and circuits, and protective sheaths for pacemakers [14]. The low mechanical stability of organosilicones limits their application as heavy load-bearing bone substitutes. However, they can be used for deposition of thin coatings onto bone implants to improve cell-contacting properties of implants’ surface. For the preparation of such coatings of great interest are organosilicones, obtained by plasma polymerization. Plasma polymerization allows deposition of high-dense, pinhole-free, and well-adherent films on a variety of substrates like conventional polymers, glass, and metals. Other advantages of the plasma polymerization process are the easy varying of process parameters and the use of modificators and fillers to produce new materials and composites with stable properties [15].

On the other side, a member of the nanocarbon family, detonation nanodiamond (DND), has emerged recently as a novel promising material for biological applications [16–22]. The nanoscale diamond material is chemically robust, nontoxic at both cellular and organism levels, and easily functionalized with different macromolecules [23–25]. Therefore, nanodiamonds can be used as reinforcements or additives in various materials to increase mechanical stability and to improve tissue interactions [26–28]. One advantage of nanoparticles as polymer additives compared to traditional additives is that the loading requirements are quite low, meaning that a small amount of nanoparticles is necessary to alter properties of materials. The inclusion of only a few percent of nanosized diamond particles into a polymer matrix may increase the stiffness and strength of the polymers and may also create nanotopographic features that mimic the nanostructure of bones. The properties of polymer–nanodiamond composites can be easily tailored by changing the type, concentration, and size of nanoparticles. However, the incorporation of particles into the polymer matrix strongly influences the bonding between particles and polymers and thus the properties of the obtained composite material [29]. Currently, there is not enough information concerning how the incorporation of nanodiamond particles into a siloxane matrix affects the surface properties of siloxane–nanodiamond composites and how this can be used to control the osteoblast and mesenchymal stem cell behavior for the purpose of bone tissue engineering.

In this study, we addressed the need for the development of methods targeted at composite layers based on plasma-polymerized hexamethyldisiloxane (PPHMDS) and DND particles as polymer modificators. The aim was to correlate the incorporation approach with physicochemical characteristics of composite layers and to characterize the cell behavior of osteoblastic cell line (MG-63) and rat mesenchymal stem cells (rMSCs). The different cell models were used in order to elucidate if the biological response to composites is more material-specific than cell-specific.

The surface roughness and the wettability of plasma-polymerized (PP) layers were evaluated by using the atomic force microscopy (AFM) and contact angle measurements. The cell adhesion was characterized by studying the overall cell morphology, cell attachment and spreading, and actin cytoskeleton organization. In addition, the cell proliferation and the alkaline phosphatase (ALP) activities of both cell types were measured. Different cell models were used to elucidate if the biological response to composites is more material-specific than cell-specific. The effect of fibronectin (FN) preadsorption of materials on cell behavior was studied.

2 Materials and Methods

2.1 Synthesis of polymer/nanodiamond composite layers

Two different composite DND/PPHMDS materials and one pure polymer, PPHMDS, were synthesized, following the method of plasma polymerization. For the preparation of polymer PPHMDS layers and the composite layers (DND/PPHMDS), hexamethyldisiloxane (HMDS; Merck, Germany) with purity > 99.99% was used. The detonation-synthesized nanodiamond powder was obtained from detonation soot, produced by SRTI-BAS (Sofia, Bulgaria), with the subsequent purification from nondiamond carbon and metal impurities through oxidation with potassium dichromate in sulfuric acid and refinement with HNO3 and HCl [30].

For the preparation of DND/PPHMDS composites, two approaches for the incorporation of DND particles into the siloxane matrix were applied: (1) the plasma deposition of preliminary prepared mixture of monomer (HMDS) and DND nanoparticles by the...