A study of the interactions between human osteoblast-like cells and polymer composites with functionalized graphene derivatives using 2D correlation spectroscopy (2D-COS)

Highlights

• Nanocomposites composed of a PCL and GO/rGO graphene derivatives were studied.

• Raman 2D-COS analysis can be used for early testing of bioactivity.

• 2D-COS revealed a complex cellular response in U-2 OS cells on the PCL/GO material.

• PCL modification with GO improves the biocompatibility of the material.

Abstract

In response to the growing need for development of modern biomaterials for applications in regenerative medicine strategies, the research presented here investigated the biological potential of two types of polymer nanocomposites. Graphene oxide (GO) and partially reduced graphene oxide (rGO) were incorporated into a poly(ε-caprolactone) (PCL) matrix, creating PCL/GO and PCL/rGO nanocomposites in the form of membranes. Proliferation of osteoblast-like cells (human U-2 OS cell line) on the surface of the studied materials confirmed their biological activity. Fluorescence microscopy was able to distinguish the different patterns of interaction between cells (depending on the type of material) after 15 days of the test run. Raman micro-spectroscopy and two-dimensional correlation spectroscopy (2D-COS) applied to Raman spectra distinguished the nature of cell-material interactions after only 8 days. Combination of these two techniques (Raman micro-spectroscopy and 2D-COS analysis) facilitated identification of a much more complex cellular response (especially from proteins) on the surface of PCL/GO. The presented approach can be regarded as a method for early study of the bioactivity of membrane materials.

Introduction

The development of nanotechnology has paved the way for novel strategies in medicine and biotechnology, making it possible for new tools and methods to be introduced into the field of medical therapy and diagnostics. Regenerative medicine and tissue engineering, in particular, have developed rapidly with the advent of nanotechnology. This progress is due to the possibility of producing biomaterials that mimic biological structures, such as nanoparticles and scaffolds for tissue engineering, as well as surface modification of previously used implant materials. The advancement of nanotechnology has created completely new possibilities for mimicking various types of extracellular matrixes [1], [2], [3], [4]. Many nanomaterials are proposed for such strategies. However, many of these still have limitations that need to be overcome. Overall, there is a continuing need to develop new nanomaterials with biological potential [5], [6].

Carbon nanostructures, such as graphene (the prime representative of such structures), carbon nanotubes, carbon nanofibers and fullerenes, etc., have a wide range of desirable properties in many modern applications. Firstly, carbon nanostructures are extremely durable, exhibit unique mechanical properties, and have above-average electrical conductivity and thermal resistance. In addition, carbon nanostructures have biocompatibility with body tissues, which is especially important when designing materials for medical applications. In particular, nanomaterials with this feature seem to be promising candidates for biomedical applications. In tissue engineering strategies, carbon nanostructures can form a composite nanoadditive or create a matrix structure that supports tissue self-regeneration [7], [8].

Graphene is a crystalline, two-dimensional structure consisting of tightly packed carbon atoms forming a hexagonal mesh with a honeycomb structure in a plane one atom thick. Graphene and its derivatives are characterized by an exceptionally large specific surface area (2630 m²/g); extraordinary mechanical properties, with a Young’s modulus equal to 1 TPa; and extremely high electrical conductivity (graphene is a semiconductor with a zero band gap) [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19].

Graphene oxide (GO) is one of the most popular graphene derivatives; unlike pure graphene, it is hydrophilic due to the presence, on its surface, of polar oxygen-containing groups [20], [21]. At the same time, a consequence of binding oxygen groups to the aromatic carbon network is disturbance of sp² hybridization of the electron orbitals of carbon atoms, leading to creation of point defects. This modification changes the properties of electrical and thermal conductivity in the plane to such an extent that the GO layer can even become an insulator [9], [15], [22], [23]. Over the years, the main reason for the interest in graphene oxide has been the possibility of reducing it to a layer of reduced graphene oxide (rGO). This process can be performed using three methods: thermal, chemical and hybrid (combination of both the previous ones). After reduction, high electrical conductivity of pure graphene is recovered, but the chemical functionality of the material is lost [14], [15], [19], [21], [22]. Another consequence of reducing the number of groups attached to the graphene sheet is strong stacking interactions between the rGO planes, and thus their agglomeration [23], [12]. In most cases, the product of GO reduction is not the ideal structure of pure graphene but partially reduced graphene oxide, on the surface of which some oxygen groups remain [24].

Graphene and its derivatives, such as GO and rGO, are widely researched for use in many branches of medicine. Potential applications include drug and gene delivery systems, and cell and tumor imaging (biosensing), particularly in cancer therapies. Graphene structures can also be used as components of miniaturized biomedical sensing devices [13], [16], [22], [25], [26], [27], [28], [29], [30], [31]. An interesting property of graphene is the possibility of antimicrobial activity, which is used in antibacterial and antiviral agents. This feature is the effect of oxidative stress and sharp edges of structures that cause disruption of the bacterial cell membrane [32], [33], [34]. Moreover, graphene and its derivatives, especially graphene oxide, are promising candidates for the role of nanofillers in biomedical materials for tissue implants and wound dressings. An additional advantage may be the antimicrobial characteristics of some graphene-based materials, of importance given that bacterial infections are a frequent consequence of implantation procedures [33]. In addition to improving the mechanical properties of such composites, especially those based on polymers, an improvement in the biocompatibility of the material is also expected [15], [25]. Studies have confirmed that these types of nanoadditives favor proliferation and differentiation of stem cells (including bone, nerve, heart and skin tissue, etc.) grown on a given nanocomposite, thus also confirming their bioactivity [13], [19], [25], [26], [29], [35], [36], [37].

In particular, in the field of bone tissue support, it is anticipated that biomaterial scaffolds will be improved by incorporating graphene and its derivatives into their structure. The research conducted so far shows that these nanostructures, especially GO, increase the osteogenic potential of composites [25], [38]. Graphene oxide, unlike unmodified graphene, is highly compatible with most polymer systems [23]. However, biocompatibility is undoubtedly related to the unique electrical properties of graphene, which change with the degree of oxidation of the material [29].

In the presented study, two types of polymer nanocomposite membranes were analyzed, produced by introducing GO and rGO nanoparticles into a poly(ε-caprolactone) (PCL) matrix (GO/PCL and rGO/PCL, respectively) (Fig. 1). PCL is a widely used semi-crystalline polymer with a long degradation time (from 6 months to 4 years), which is non-toxic and easy to mix with other materials [39], [40], [41], [42], [43], [44]. The analyzed nanocomposite membranes were subjected to biological tests involving culturing cells of the U-2 OS human osteosarcoma line for a period of 15 days. The U-2 OS cell line is widely used in biomedical research to model osteoblast behavior [45], [46], [47]. The methods used to monitor the culture were optical and fluorescence microscopy. The Raman spectroscopy technique was also selected as a method for detecting the signal from the cell-membrane interface, where the cell adhered to the material surface. The interactions of appropriate functional groups of the nanocomposite with the osteoblast-like cells were identified by applying two-dimensional correlation spectroscopy (2D-COS) to Raman spectra collected on selected days of cell culture. The correlation method turned out to be the optimal analytical technique to distinguish the interactions of the three components of the tested complex system, consisting of a polymer matrix, a carbon nanoadditive and osteoblast-like cells.

The aim of the study presented here was to evaluate the behavior of cells on the surface of the studied polymer nanocomposites in vitro, and thus check the ability of the prepared materials to act as a substrate for cell growth in vivo. The 2D-COS method presented in the paper for assessing the bioactivity of nanocomposite materials facilitates early identification of changes occurring at the interface between the cell and the biomaterial.