Combinatorial MAPLE deposition of antimicrobial orthopedic maps fabricated from chitosan and biomimetic apatite powders

https://doi.org/10.1016/j.ijpharm.2016.07.015Get rights and content

Highlights

  • Blended chitosan-to-biomimetic apatite films deposited by combinatorial MAPLE.

  • Amorphous thin films with rough morphology.

  • Composition gradient of films monitored by FTIR and XPS, confirmed by Raman.

  • Antimicrobial activity controlled by chitosan content of films.

Abstract

Chitosan/biomimetic apatite thin films were grown in mild conditions of temperature and pressure by Combinatorial Matrix-Assisted Pulsed Laser Evaporation on Ti, Si or glass substrates. Compositional gradients were obtained by simultaneous laser vaporization of the two distinct material targets. A KrF* excimer (λ = 248 nm, ϿFWHM = 25 ns) laser source was used in all experiments. The nature and surface composition of deposited materials and the spatial distribution of constituents were studied by SEM, EDS, AFM, GIXRD, FTIR, micro-Raman, and XPS. The antimicrobial efficiency of the chitosan/biomimetic apatite layers against Staphylococcus aureus and Escherichia coli strains was interrogated by viable cell count assay.

The obtained thin films were XRD amorphous and exhibited a morphology characteristic to the laser deposited structures composed of nanometric round shaped grains. The surface roughness has progressively increased with chitosan concentration. FTIR, EDS and XPS analyses indicated that the composition of the BmAp-CHT C-MAPLE composite films gradually modified from pure apatite to chitosan.

The bioevaluation tests indicated that S. aureus biofilm is more susceptible to the action of chitosan-rich areas of the films, whilst the E. coli biofilm proved more sensible to areas containing less chitosan.

The best compromise should therefore go, in our opinion, to zones with intermediate-to-high chitosan concentration which can assure a large spectrum of antimicrobial protection concomitantly with a significant enhancement of osseointegration, favored by the presence of biomimetic hydroxyapatite.

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 France⿿Romania 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

References (80)

  • D. Grossin et al.

    Biomimetic apatite sintered at very low temperature by spark plasma sintering: physico-chemistry and microstructure aspects

    Acta Biomater.

    (2010)
  • A.M. Grumezescu et al.

    Synthesis, characterization and in vitro assessment of the magnetic chitosan-carboxymethylcellulose biocomposite interactions with the prokaryotic and eukaryotic cells

    Int. J. Pharm.

    (2012)
  • I.M. Helander et al.

    Chitosan disrupts the barrier properties of the outer membrane of Gram⿿negative bacteria

    Int. J. Food Microbiol.

    (2001)
  • Q. Hu et al.

    Preparation and characterization of biodegradable chitosan/hydroxyapatite nanocomposite rods via in situ hybridization: a potential material as internal fixation of bone fracture

    Biomaterials

    (2004)
  • L.N. Jia et al.

    Morphology and composition of coatings based on hydroxyapatite-chitosan-RuCl3 system on AZ91D prepared by pulsed electrochemical deposition

    J. Alloy. Compd.

    (2016)
  • Z. Jia et al.

    Bioinspired anchoring AgNPs onto micro-nanoporous TiO2 orthopedic coatings: trap-killing of bacteria, surface-regulated osteoblast functions and host responses

    Biomaterials

    (2016)
  • M. Kong et al.

    Antimicrobial properties of chitosan and mode of action: a state of the art review

    Int. J. Food Microbiol.

    (2010)
  • I. Leceta et al.

    Characterization and antimicrobial analysis of chitosan-based films

    J. Food Eng.

    (2013)
  • M. Lee et al.

    Biomimetic apatite-coated alginate/chitosan microparticles as osteogenic protein carriers

    Biomaterials

    (2009)
  • X. Li et al.

    H. Chen Preparation and characterization of nano-hydroxyapatite/chitosan cross-linking composite membrane intended for tissue engineering

    Int. J. Biol. Macromol.

    (2012)
  • X. Li et al.

    Osseointegration of chitosan coated porous titanium alloy implant by reactive oxygen species-mediated activation of the PI3 K/AKT pathway under diabetic conditions

    Biomaterials

    (2015)
  • X.Y. Ma et al.

    The promotion of osteointegration under diabetic conditions using chitosan/hydroxyapatite composite coating on porous titanium surfaces

    Biomaterials

    (2014)
  • N. Mihailescu et al.

    I.N. Mihailescu Structural, compositional, mechanical characterization and biological assessment of bovine-derived hydroxyapatite coatings reinforced with MgF2 or MgO for implants functionalization

    Mater. Sci. Eng. C

    (2016)
  • H.K. No et al.

    Antibacterial activity of chitosans and chitosan oligomers with different molecular weights

    Int. J. Food Microbiol.

    (2002)
  • J. Peña et al.

    Room temperature synthesis of chitosan/apatite powders and coatings

    J. Eur. Ceram. Soc.

    (2006)
  • P. Sendi et al.

    Periprosthetic joint infection following Staphylococcus aureus bacteremia

    J. Infect.

    (2011)
  • F. Sima et al.

    Combinatorial Matrix Assisted Pulsed Laser Evaporation of a biodegradable polymer and fibronectin for protein immobilization and controlled release

    Appl. Surf. Sci.

    (2014)
  • A. Simchi et al.

    Recent progress in inorganic and composite coatings with bactericidal capability for orthopaedic applications

    Nanomed.-Nanotechnol. Biol. Med.

    (2011)
  • M. Sivakumar et al.

    Preparation, characterization and in-vitro release of gentamicin from coralline hydroxyapatite-chitosan composite microspheres

    Carbohydr. Polym.

    (2002)
  • I.A. Sogias et al.

    Chitosan-based mucoadhesive tablets for oral delivery of ibuprofen

    Int. J. Pharm.

    (2012)
  • L. Song et al.

    Antibacterial hydroxyapatite/chitosan complex coatings with superior osteoblastic cell response

    Mater. Lett.

    (2011)
  • R.A. Surmenev

    A review of plasma-assisted methods for calcium phosphate-based coatings fabrication

    Surf. Coat. Technol.

    (2012)
  • W.W. Thein-Han et al.

    Biomimetic chitosan⿿nanohydroxyapatite composite scaffolds for bone tissue engineering

    Acta Biomater.

    (2009)
  • A. Visan et al.

    Biomimetic nanocrystalline apatite coatings synthesized by Matrix Assisted Pulsed Laser Evaporation for medical applications

    Mater. Sci. Eng. B

    (2014)
  • D. Vllasaliu et al.

    Absorption-promoting effects of chitosan in airway and intestinal cell lines: a comparative study

    Int. J. Pharm.

    (2012)
  • Q. Yuan et al.

    Controlled and extended drug release behavior of chitosan-based nanoparticle carrier

    Acta Biomater.

    (2010)
  • C.R. Arciola et al.

    Biofilm-based implant infections in orthopaedics

    Adv. Exp. Med. Biol.

    (2015)
  • E. Axente et al.

    Combinatorial MAPLE gradient thin film assemblies signalling to human osteoblasts

    Biofabrication

    (2014)
  • B.D. Boyan et al.

    Effect of surface roughness and composition on costochondral chondrocytes is dependent on cell maturation state

    J. Orthop. Res.

    (1999)
  • H.I. Chang et al.

    Cell responses to surface and architecture of tissue engineering scaffolds

  • Cited by (20)

    • Antimicrobial coatings based on chitosan to prevent implant-associated infections: A systematic review

      2021, iScience
      Citation Excerpt :

      The produced coatings decreased the number of biofilm cells up to 3 Log and biofilm metabolic activity by 50% (Shi et al., 2016). In addition, Visan et al. (2016) blended CS to biomimetic apatite films, which were deposited by the combinatorial matrix-assisted pulsed laser evaporation method on titanium discs and investigated their efficacy to prevent E. coli and S. aureus biofilm formation on bone implants. Apatite-CS surfaces reduced biofilms viability by 1 and 2 Log for E. coli and S. aureus, respectively, after 48 h of exposure (Visan et al., 2016).

    • Effects of native oxidation on Ti/TiO<inf>2</inf> nanodot arrays and their plasmonic properties compared to Au nanodot arrays

      2021, Applied Surface Science
      Citation Excerpt :

      The small peaks of the Ti/TiO2 film on the ITO glass at 37.5° and 63.3° exhibited XRD patterns corresponding to pure titanium (ICDD:00–044-1294) [42]. A small peak at 41.6° was associated with a titanium sub-oxide phase (TiO) (ICDD:01–086-2352) [43]. The Ti/TiO2 film on the ITO glass exhibited a peak at 55.6° associated with hydrogenated rutile TiO2 and an amorphous phase (JPCDS:00–021-1272,00–002-0406) [42].

    • Gradient multifunctional biopolymer thin film assemblies synthesized by combinatorial MAPLE

      2019, Applied Surface Science
      Citation Excerpt :

      FTIR spectrum of QCH (Fig. 2) revealed the presence of all characteristic bands. This is supported by the broad OH and sharp NH absorption peaks of CH at 3400 and 3100 cm−1, and CH bond in CH2 (ʋ1 = 2920 cm−1) and CH3 (ʋ2 = 2875 cm−1) groups, respectively [58]. Peaks observed in the range of 1200–1000 cm−1 are assigned to the COC bending vibration of glucose rings and glucose amine bonds, proving that the main chain of CH was not decomposed by oxidation and quaternization.

    • Antimicrobial Thin Coatings Prepared by Laser Processing

      2017, Nanostructures for Antimicrobial Therapy: Nanostructures in Therapeutic Medicine Series
    View all citing articles on Scopus
    View full text