Fluor‑carbonated hydroxyapatite coatings by pulsed laser deposition to promote cell viability and antibacterial properties
Introduction
Fluorine (F) is an essential element present in bone and dental tissues. It is thought to be an enhancer of the synthesis of bone cell growth factors, acting primarily on the osteoprogenitor cells and/or undifferentiated osteoblasts cells rather than on highly differentiated osteoblasts. It is therefore related to the promotion of mineralization and is directly involved in the bone formation process [1]. Fluorine contributes to bone healing and regeneration by inducing the differentiation of osteoprogenitor and undifferentiated precursor cells into osteoblasts [2]. Moreover, in the form of fluorapatite, it contributes to the development of a highly crystalline and stable apatite [3], with improved mechanical properties in relation to geological hydroxyapatite, with good stiffness, high elastic modulus and hardness.
The antibacterial effects of fluorine in the form of fluoride on oral bacteria are also well known [4]. There is evidence that fluoride can interfere with enzyme activity and reduce acid production by oral bacteria (both Gram-positive and Gram-negative), thereby inhibiting the enrichment of cariogenic species within dental plaque [5, 6]. The capacity of fluorine (released from a cement) to inhibit the metabolism of certain bacteria (i.e., oral streptococci) has previously been reported [7]. Studies suggest that fluoride also has anti-plaque properties. It is well documented that amine fluoride and stannous fluoride possess bactericidal properties against oral bacteria [8, 9]. The pre-incubation of hydroxyapatite with amine fluoride significantly decreases the viability of Streptococcus sobrinus in biofilm, whereas sodium fluoride or chlorhexidine does not [10].
Joint prostheses are one of the most important medical advances made to date due to their ability to relieve joint pain, restore joint function, and allow millions of patients around the world to recover their independence [11]. However, these prostheses are liable to failure, the main causes being aseptic loosening, infection, dislocation, and fracture of the prosthesis or bone [12]. Aseptic loosening in particular can occur through loss of fixation caused by a lack of initial tissue-implant fixation, leading to a mechanical loss over time and/or particle-induced osteolysis around the implant [13]. Therefore, if osteointegration were promoted, aseptic loosening could be prevented [14].
Osteointegration can be promoted by covering prostheses with a calcium phosphate layer, mimicking the inorganic composition of bone [14]. Applying such a layer to the surface of titanium implant devices has been proven to enhance bone formation around the implants and contribute to cementless fixation, improving clinical success at an early stage after implantation [15]. Plasma-spray was the first technique used to produce calcium phosphate coatings on dental root implants and hip and knee orthopedic prostheses. However, the high thickness of the coatings (50–200 μm, limitation of the technique) resulted in their partial disappearance during the implantation time, causing the proliferation of macrophages. The pulsed laser deposition (PLD) technique, on the other hand, produces thin coatings with good adhesion properties; it also allows the precise control of the chemistry and crystallinity of the coatings [16, 17]. Synthetic carbonated hydroxyapatite (HA) coatings produced by PLD have been successfully tested, showing enhanced or similar osteointegration to their plasma-sprayed equivalents, but with improved adhesion properties, without the risk of delamination or detachment of the coating [[18], [19], [20], [21], [22], [23], [24], [25]].
Prosthetic joint infection occurs infrequently (1–2% of all cases), though it is a devastating complication with high morbidity and substantial costs. The economic burden of prosthetic joint infection is expected to grow in the coming years due to the increase in patients undergoing arthroplasty replacements [26]. Gram-positive cocci, the most common of which are Staphylococcus aureus and Staphylococcus epidermidis, represent up to 77% of all infections [[27], [28], [29], [30], [31]].
Apart from the administration of oral or intravenous antibiotics to prevent bacterial colonization, the local action of different chemical elements incorporated in trace concentrations into the calcium phosphate structure has been investigated. HA coatings doped with Se, Ag, Cu or Zn have been proven to reduce bacterial biofilm formation due to the chemical changes they promote in the cellular environment [18, [20], [21], [22], [23], 32].
Given the interesting properties of fluorine, this paper will describe the use of PLD to coat metallic implants with a thin layer of bioceramic of marine origin, mainly composed of fluorapatite obtained from the enameloid of shark teeth. It will evaluate the structure and composition of different coatings obtained at various H2O vapor pressures. The vapor pressure exercise examines the dependence of the coatings on pressure, to guarantee optimal properties and good transference from the original bioceramic. Finally, the proliferation and alkaline phosphatase (ALP) synthesis of MC3T3-E1 pre-osteoblasts on the fluor‑carbonated HA coatings, together with their antibacterial properties against Staphylococcus aureus and Staphylococcus epidermidis strains, will be discussed.
Section snippets
Coating process
Following the same methodology as previous studies [[33], [34], [35], [36]], the fluor‑carbonated HA coatings were processed using a UV ArF* excimer laser (λ = 193 nm) source (Lambda Physik COMPEX 205). PLD targets with a mass of 2 g and a diameter of 20 mm were prepared using a bioceramic, developed in our laboratories, obtained from the enameloid of shark teeth (Isurus oxyrinchus and Prionace glauca species) [37, 38]. The bioceramic granulate was compacted by raising the pressure to 8 tons
Coating characterization: dependence on H2O vapor pressure
The FTIR spectra of the Bio-FHA coatings deposited at H2O vapor pressures of 0.45 and 0.0 mbar are presented in Fig. 1. The main vibration modes were identified using previous literature [24, [46], [47], [48], [49], [50], [51], [52], [53]] as follows: (i) phosphate groups with a strong peak located between 1000 and 1200 cm−1 attributed to the asymmetric stretching of PO bonds, a weak band at 950 cm−1 due to symmetric stretching and an absorption band between 550 and 600 cm−1 attributed to
Conclusions
This study demonstrated both the feasibility of producing PLD coatings (Bio-FHA) from shark tooth enameloid in a H2O vapor atmosphere and good compositional transference from the original bioceramic. It also showed how the coatings' composition and crystalline structure depended on the H2O vapor pressures applied during the process. Increased pressure meant increased crystallinity: at the highest pressure of 0.45 mbar, more defined bands attributed to apatite diffraction planes, and crystallite
Acknowledgements
This research was partially supported by 0245 IBEROS1E and 0302 CVMARI1P, both from the INTERREG V Spain-Portugal (POCTEP) program; and Competitive Reference Groups (GRC) ED431C 2017_51 and Research networks ED431D 2017/13 both from the Xunta de Galicia, Spain. Hidalgo-Robatto B. M. acknowledges funding support from Xunta de Galicia pre-doctoral grant (A-2014). Technical support from CACTI (Universidade de Vigo) and J. Dorado are also gratefully acknowledged.
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