Fluor‑carbonated hydroxyapatite coatings by pulsed laser deposition to promote cell viability and antibacterial properties

Highlights

• The role of H₂O vapor pressure in the F‑carbonated-HA coating properties is explored.

• Hydrated F‑carbonated-HA coating with 1.96 F at.% promoted antibacterial properties.

• Biofilm of most common bacteria in prosthetic infections was reduced (p < 0.05).

• MC3T3-E1 pre-osteoblasts differentiate (p < 0.01) on hydrated F‑carbonated-HA coatings.

Abstract

Fluorine, an essential element present in bone and dental tissues, promotes mineralization and is directly involved in the bone formation process. The antibacterial effects of fluorine on oral bacteria are also well known. In this study, metallic implants were coated with a thin layer of calcium phosphate enriched with fluorine to improve osteointegration and protect against infections. These coatings were obtained by pulsed laser deposition (PLD) using, as a target, a bioceramic of marine origin, mainly composed of fluorapatite from the enameloid of shark teeth. The compositional dependence of coatings on H₂O vapor pressures applied during PLD was analyzed in order to optimize physicochemical properties. Physicochemical characterization to evaluate morphology (SEM), thickness (interferometric profilometry), structure (XRD) and composition (FTIR, XPS) was performed. To evaluate the biological response, both MC3T3-E1 pre-osteoblasts and bacterial strains Staphylococcus aureus and Staphylococcus epidermidis, most responsible for 77% of infections associated with prosthetic implants, were tested. Proper cell proliferation (MTT assay) and ALP synthesis up to 21 days were confirmed. Antibacterial properties were also demonstrated: compared to synthetic hydroxyapatite coatings, there was a significant reduction in colony-forming units (CFUs) for both strains.

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.