A 2D-Raman correlation spectroscopy study of the interaction of the polymer nanocomposites with carbon nanotubes and human osteoblast-like cells interface
Graphical abstract
Introduction
Tissue engineering medicine emerged as an interdisciplinary field of study years ago as an answer to the enormous demand of biomedical substitutes that could replace, maintain or even improve damaged tissue functions.
Even though autografts as well as allografts are regarded as conventional tissue replacements, to the extent that autografts are still called the best clinical standards, both suffer from plenty of flaws connected with their low availability, immune rejections and overall high failure rate [[1], [2], [3]]. Tissue engineering aims to overcome those problems by introducing new and advanced biomaterials that can be designed to construct artificial tissues and organs [4]. Ideal substitutes should not only provide mechanical support in a place of tissue shortage but also positively interact with cells, integrate with host cells and even actively regulate tissue regeneration [1]. In order to develop biomaterials that could fulfill all those requirements tissue engineering needs to utilize principles of both life science and engineering, that is what made this field so multidisciplinary and complex [5]. Since materials science, especially nanotechnology is a rapidly growing field of science, it is believed that tissue engineering is about to make a giant step with its convergence with nanotechnological strategies [1].
Nanomaterials were proven to possess superior physiochemical properties to conventional materials for tissue growth [5]. Furthermore, because the size of a nanoparticle is comparable to the dimention of natural entities (such as collagen fibres) it can mimic tissue surface properties and even be involved in biological processes [6,7]. Nowadays, the usage of nanoparticles as a modifiers of polymeric materials to form so-called polymer-based nanocomposites brings great attention [3]. The incorporation of nano-size particles into polymer structure modify polymeric matrix at nanoscale, therefore closely to the molecular level leading to the improvement of mechanical, electronic, biocompatible properties and even the emerging of new surprising properties of the material [3,8].
Among lots of current tissue engineering challenges one of the most urgent is the creation of scaffold for bone tissue shortage treatment [9]. Basically, scaffold serves as a template and structural support for bone stem cells migration, adhesion, proliferation and differentiation so overall scaffold supports osteoblasts growth and supplies an appropriate microenvironment for the newly formed tissue [4,10,11]. Therefore the biomaterial used to design such scaffold should possess a number of properties that are not easy to achieve all together and it is a reason why there is a constant need of developing new materials that can face all of those requirements. First of all, the candidate for bone scaffold should be biocompatibile with bone tissue-specific cell types and with other environments elements, such as body fluids [4]. Also it needs to be able to bear external force - possess sufficient mechanical properties, optimal porosity, be osteoconductive and osteoinductive as well as be biodegradable with time of resorption synchronized with replacement by natural tissue growth (the mechanical support must be served until new tissue is able to support itself, in the case of bone scaffold it should last 12 weeks at least) [2]. Due to many drawbacks of naturally derived polymers, the synthetic polymers are regarded as suitable candidates for bone tissue engineering [1]. Among them, poly(ε-caprolactone) (PCL) seems to be almost ideal basic material for scaffold owning to its longer time of degradation than other synthetic polymers, and also this time is adequate for bone tissue regeneration [9,11]. PCL is a semi-crystalline poly(α-hydroxy) acid (Fig. 1) that is non-toxic, cyto-compatible, and has an excellent rheological and viscoelastic properties that makes it easy to blend with other polymers and additives allowing its modification to obtain preferred properties [10,11]. The last assesment is extremely important taking into consideration the fact that raw PCL does not meet the optimal term of biocompatibility (because of its highly hydrophobic nature) and mechanical durability. However, the PCL has a tremendous potential for bone tissue engineering and strategies of its modification in order to improve biocompatibility are in huge demand [10]. The utilization of nanotechnology accomplishments can be a successful way to create an optimal PCL nanocomposite serving as bone scaffold.
The discovery of carbon nanotubes in 1991 by Iijima was a landmark achievement in the field of nanotechnology and since then CNTs are the subject of researchers’ undiminished interest because of their unique properties [12]. Carbon nanotubes are formed by graphene sheets rolled into hollow cylinders and two types of CNTs are possible to obtain: single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) [4,13,14]. This allotrope form of carbon is proven to possess extraordinary properties such us: ultrahigh ‘surface area to volume’ ratio, outstanding mechanical strength (one of the strongest material ever studied), high electrical and thermal conductivity, stiffness, ultra-light weight and chemical and thermal stability [3,4,13,15,16]. The general biocompatibility of CNTs is still being under intense research, however, so far studies have already shown that CNTs in low concentrations are not toxic for human osteoblasts [7]. More significantly – it was proven that CNTs used as additives in scaffold can even improve cell adhesion and therefore support their growth on scaffold [3,13]. Taking into consideration all of the aforementioned carbon nanotubes characteristics, the potential of CNTs as a reinforcement material in polymer nanocomposites for bone tissue engineering applications is evident. Since the pristine CNTs tend to agglomerate in aqueous environmental or even to reagglomerate in polymeric matrix, the strategies to improve dispersion and its stabilization are implemented to the process of nanocomposite production and these strategies include sonification, adding small quantities of nanoadditives and prior functionalization of the nanoparticles [17]. The functionalization procedure is connected with the chemical modification of CNTs’ side walls surfaces that involves the creation of covalent linkage with functional groups, usually by oxidation. This method is believed to not only enhance the dispersion of nanoparticles, but additionally ensure better interactions between CNTs and polymer as well as an overall improvement of material biocompatibility [16].
In the presented research the polymer nanocomposites were produced through the incorporating of multi-walled carbon nanotubes (in the raw state and after oxidative functionalization, respectively) into a poly(є-caprolactone) matrix (the chemical structure of PCL is presented in Fig. 1). The obtained carbon nanocomposites in the form of membranes were subjected to biological test. The common model used for osteoblast in biomedical research are osteoblast-like U-2 OS cells (human bone osteosarcoma epithelial cells) [18,19]. The U-2 OS cells were cultured on the surface of the nanocomposite samples for 8 days. On the 1st, 3rd, 6th, and 8th day Raman spectra were measured so the interface of cell and material was monitored. The Raman spectroscopy was used for the reason that it is an extremely valuable technique to test short-range structure ordering and it is widely used for characterisation of the polymeric and carbonaceous materials [20]. In our previous research this technique was successfully applied to determine the decrease of the crystallinity level in the polymeric matrix after the incorporation of the carbon nanotubes into it [21]. However, the Raman bands intensity of the studied material is much higher than that of the cells therefore, the 2D Raman correlation spectroscopy was used to control changes in the structure of the nanocomposites and the interaction with the osteoblast-like cells in order to decode the relations hidden in the Raman spectra. In this mathematical analysis the day of measurement was regarded as an external perturbation.
Section snippets
MCWCNTs and their functionalization
The multi-walled carbon nanotubes, MWCNTs (Nanostructured & Amorphous Materials, USA), were subjected to the functionalization process in order to create oxide groups on their surface. The MWCNT were purified and immersed into a mixture of sulphuric (VI) acid and 65% nitric (V) acid (3/1 ratio) for 2 h at the temperature of 70 °C. Then, the carbon nanotubes were rinsed with distilled water and centrifuged. They were denoted as MWCNTs and the functionalized ones as MWCNTs-f.
The preparation of the nanocomposite membranes
Poly(є-caprolactone)
The proliferation test
The main purpose of MWCNTs’ incorporation into the PCL polymeric matrix was the improvement of its biocompatibility. In order to assess the characteristics of the obtained polymer nanocomposites for the bone tissue engineering applications, the study of cell viability and proliferation on these materials were evaluated. Human osteoblast-like U-2 OS-Green cells (with a stable expression of green fluorescence protein) were cultured on the tested samples: PCL/MWCNTs and PCL/MWCNTs-f in the form of
Conclusions
The results of the performed analysis revealed that the nature of the interactions between osteoblast-like U-2 OS cells and polymer nanocomposites differs in regard to the type of nanofiller introduced into the polymeric matrix. Both PCL/MWCNTs and PCL/MWCNTs-f were confirmed to be biocompatible and osteoinductive by the performed cell proliferayion test. The identification of the specific cells-biomaterial interactions was possible by the usage of the 2D Raman correlation spectroscopy
CRediT authorship contribution statement
Anna Kołodziej: Methodology, Investigation, Visualization, Writing - original draft. Aleksandra Wesełucha-Birczyńska: Conceptualization, Formal analysis, Writing - review & editing. Małgorzata Świętek: Methodology, Investigation. Łukasz Skalniak: Methodology, Visualization. Marta Błażewicz: Conceptualization.
Declaration of competing interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This project was financed from the National Science Centre (NCN, Poland) on the decision 2013/09/B/ST8/00146. AK has been partly supported by the EU Project POWR.03.02.00–00-I004/16.
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