Combinatorial MAPLE deposition of antimicrobial orthopedic maps fabricated from chitosan and biomimetic apatite powders
Graphical abstract
Introduction
Natural polymers are currently employed to obtain tailored systems for drug passive/active targeting in order to decrease the incidence of the side effects (Kim et al., 2008). The natural polymers exhibit the major advantage of biodegradability inside the human body, not requiring removal or additional manipulation (Nair and Laurencin, 2006). Due to their excellent biocompatibility and cost-effectiveness they can be used as pharmaceutical excipients (Chifiriuc et al., 2014).
Among natural polymers used for drug delivery, chitosan (CHT) is a highly biodegradable, non-toxic and biocompatible cationic polysaccharide synthesized from chitin by alkaline deacetylation (Vllasaliu et al., 2012, Sogias et al., 2012, Derakhshandeh and Fathi, 2012, Gan and Wang, 2007). The chitosan potential to be used as an antimicrobial agent (Martins et al., 2014), was stressed upon together with delivery of antibiotics, such as beta-lactams (e.g., penicillins, cephalosporins), aminoglycosides and daptomycin (Noel et al., 2008, Grumezescu et al., 2012, Grumezescu et al., 2011).
Nevertheless, CHT application in anti-infective strategies is limited because of its low solubility at neutral or basic pH. For this reason, the focus has been recently moved to the design of antimicrobial films consisting of CHT and its derivatives. They demonstrated excellent inhibitory activity against a wide spectrum of Gram-positive and Gram-negative bacteria, including food contaminants (Kong et al., 2010, Dutta et al., 2009, Leceta et al., 2013, Dai et al., 2011).
Recent studies revealed the potential of CHT/hydroxyapatite (HA) composites to be used as coatings on titanium surface in order to increase the osseointegration capacity of bone implants (Ma et al., 2014, Li et al., 2015). There exist however, a few studies only, aiming to evaluate the anti-biofilm activity of such coatings, an aspect of key importance for the prevention and control of implant-associated infections.
Many authors have reported the preparation of mixtures of calcium phosphates (CaP) and CHT in the form of powders (Yoshida et al., 2004), membranes (Ito et al., 1999), scaffolds (Zhang and Zhang, 2002), or microspheres (Sivakumar et al., 2002). Nevertheless, only few publications were focused on developing procedures allowing the concomitant preparation of a composite material containing the two components, which is expected to ensure a more intimate contact between them (Hu et al., 2004, Davidenko et al., 2010, Thein-Han and Misra, 2009).
CHT-CaP composite films have been synthesized by several methods like: pulsed electrochemical deposition (Jia et al., 2016a), electrophoretic deposition (Zhong et al., 2015), plasma spraying (Song et al., 2011), or co-precipitation method (Peña et al., 2006).
On the other hand, over the past decades, laser techniques proved a high potential for the fabrication of antimicrobial coatings for orthopedic implants for medical applications such as the bone tissue replacement or treatment of osteoporosis or osteolytic tumors (Jia et al., 2016b, Simchi et al., 2011).
Furthermore, the laser-based technologies are exhibiting a lot of advantages, as they allow for the fabrication of a wide-range of different biomaterials, with a fairly uniform spreading of material over rather large areas, controlled film thickness (with an accuracy of 1 ÿ), good adhesion to substrate (Blind et al., 2005, Duta et al., 2013, Mihailescu et al., 2016), and specific surface properties (Sima and Mihailescu, 2013). Moreover, these deposition methods imply a low material consumption, and ensure the stoichiometry preservation of the growing films (Eason, 2006, Yu et al., 2014, Cristescu et al., 2012). In the same time, remarkable efforts were recently paid to the development by combinatorial processing of new biomaterials with innovative properties. Usually, the fabrication of a composite layer is carried out by premixing of biopolymer solutions followed by heating of coating (Meredith et al., 2000) or film casting/solvent evaporation (Li et al., 2012). The combinatorial technology for the blending of two different biomaterials (Torricelli et al., 2015, Sima et al., 2014, Axente et al., 2014, Sima et al., 2012) is based on Matrix-Assisted Pulsed Laser Evaporation (MAPLE) method. This newly developed technique Combinatorial-MAPLE (C-MAPLE) stands for a simple, single step, fabrication route which can easily limit the time of manipulation and biomaterials consumption.
The aim of this study was to synthesize thin coatings containing natural biopolymer chitosan combined with biomimetic apatite (Eichert et al., 2007, Grossin et al., 2010, Visan et al., 2014) directly on titanium implants by C-MAPLE technique.
As known, a biomaterial used for bone substitution should possess a set of ineluctable properties. They are:
- (i)
An identical chemical composition to the natural bone (which is a complex structure of organic and inorganic materials (Dorozhkin, 2009)). For this purpose, the nonstoichiometric biomimetic apatite (BmAp) (Eichert et al., 2007, Grossin et al., 2010, Visan et al., 2014), has been used as model for the basic constituent of the inorganic part of the bone, and chitosan (CHT), a natural biopolymer, with a similar chemical structure to the glycosaminoglycan, the prevalent extracellular matrix of the bone and cartilage, as the organic phase of bone (Vllasaliu et al., 2012).
- (ii)
A good mechanical strength. The coatings were therefore deposited on titanium (Ti) medical implants, thus harmoniously combing the excellent mechanical features of Ti with the biomimetics of the organic-inorganic biofunctional layers (Agarwal and García, 2015). This will confer stability and reliability to the medical device assembly.
- (iii)
Biocompatibility. From this point of view, both CHT and BmAp exhibit excellent cytocompatibility and remarkable osteoconductive properties, respectively (Song et al., 2011, VandeVord et al., 2002).
- (iv)
Resistance to microbial colonization, particularly during the osseointegration period. In this regard, CHT shows a higher antibacterial activity against a broad spectrum of microbial agents (Lee et al., 2009). It is therefore expected that the composite structures of polymer (CHT)/ceramic (BmAp) will exhibit a double function: antimicrobial protection and enhancement of osteoblast cell proliferation, opening new promising opportunities for developing a new generation of orthopedic implants.
To the best of our knowledge, this is the first attempt to synthesize a composition gradient between CHT and BmAp by laser co-evaporation of the two distinct cryogenic targets followed by a co-deposition process.
Section snippets
Materials
CHT with a low molecular weight was purchased from Sigma-Aldrich, while the biomimetic apatite powder, with a particle size <25 μm, was prepared by the co-precipitation method in accordance with a previously described protocol (Visan et al., 2014). Solutions consisting of 2% CHT and 1% BmAp in deionized water were prepared. All target solutions were poured into a copper target holder, pre-cooled at 173 K, and subsequently frozen by immersion in liquid nitrogen for 15 min.
C-MAPLE deposition process
C-MAPLE technique was used
SEM, AFM, and EDS observations
The optimal cellular response (cellular adhesion, spreading, and proliferation) is of great significance for tissue and medical engineering and is dependent on surface morphology and composition (Surmenev, 2012, Spencer, 2011).
The general surface morphology of the CHT-BmAp film has been first investigated by SEM. Fig. 2 displays the characteristic topological features of the C-MAPLE film in various surface regions of interest: far-most CHT rich region (S1), CHT-BmAp blended regions (with CHT
Conclusions
The synthesized CHT-BmAp blended thin films are amorphous, rough, with a morphology characteristic to MAPLE structures. The composition gradient of the chitosan-to-biomimetic hydroxyapatite has been confirmed longwise the combinatorial films.
The antimicrobial activity was controlled by the concentration of chitosan, while the blended structures were better integrated for quasi-equal presence of the two compounds (i.e., chitosan and biomimetic apatite). The most efficient antimicrobial activity,
Acknowledgements
The authors acknowledge the financial support of UEFISCDI under the FranceRomania bilateral contract 778/2014, the Ministère des Affaires étrangères under the PHC BRANCUSI 2015 (N° 32648SD) and the National Authority for Scientific Research and Innovation in the frame of Nucleus Programme contract 4N/2016. The authors thank to Iuliana Urzica for performing the profilometry thickness measurements. G.E.S. acknowledges with thanks the support of NIMP Core Programme PN 16 48-3/2016. The
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