A natural biogenic fluorapatite as a new biomaterial for orthopedics and dentistry: antibacterial activity of lingula seashell and its use for nanostructured biomimetic coatings

Abstract

Calcium phosphates are widely studied in orthopedics and dentistry, to obtain biomimetic and antibacterial implants. However, the multi-substituted composition of mineralized tissues is not fully reproducible from synthetic procedures. Here, for the first time, we investigate the possible use of a natural, fluorapatite-based material, i.e., Lingula anatina seashell, resembling the composition of bone and enamel, as a biomaterial source for orthopedics and dentistry. Indeed, thanks to its unique mineralization process and conditions, L. anatina seashell is among the few natural apatite-based shells, and naturally contains ions having possible antibacterial efficacy, i.e., fluorine and zinc. After characterization, we explore its deposition by ionized jet deposition (IJD), to obtain nanostructured coatings for implantable devices. For the first time, we demonstrate that L. anatina seashells have strong antibacterial properties. Indeed, they significantly inhibit planktonic growth and cell adhesion of both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. The two strains show different susceptibility to the mineral and organic parts of the seashells, the first being more susceptible to zinc and fluorine in the mineral part, and the second to the organic (chitin-based) component. Upon deposition by IJD, all films exhibit a nanostructured morphology and sub-micrometric thickness. The multi-doped, complex composition of the target is maintained in the coating, demonstrating the feasibility of deposition of coatings starting from biogenic precursors (seashells). In conclusion, Lingula seashell-based coatings are non-cytotoxic with strong antimicrobial capability, especially against Gram-positive strains, consistently with their higher susceptibility to fluorine and zinc. Importantly, these properties are improved compared to synthetic fluorapatite, showing that the films are promising for antimicrobial applications.

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1. Introduction

Calcium phosphate (CaP)-based biomaterials have been widely studied for the development of bone substitutes and implant coatings to promote osseointegration and regeneration of native bone.^1–8 The widespread use of CaPs derives from their similarity to the mineral phase of bone, since, to boost osseointegration, a composition resembling as closely as possible that of mineralized tissues is desired.^9,10 However, together with promoting integration, implantable materials shall discourage infection, which is the main reason for implant failure in orthopedics and dentistry, and leads to severe complications.^11–13 The need for antibacterial solutions is becoming increasingly important, as the progressive development of micro- and nano-texturing and of custom-made porous 3D printed implants, aiming at creating multi-scale topographical cues to promote cell attachment, proliferation and regeneration, also tends to favor bacterial adhesion. To this aim, functionalization of CaPs with antibacterial metals and/or drugs has been proposed.^14–19

However, mineralized tissues are composed of multi-substituted calcium phosphates that cannot be reproduced from synthetic processes.^20,21 More specifically, bone, enamel and dentine apatite contain substitutions in the crystal lattice by carbonate, magnesium, fluorine, strontium, sodium, chlorine, potassium, silicates, zinc and manganese ions, in variable concentrations. Each trace ion has an impact on the characteristics of the crystal lattice, affecting the solubility and crystallinity of the material, and its interactions with the host.^9,20,25–43 In addition, some ions have specific therapeutic functions, including a concentration-dependent capability to inhibit microbial proliferation.^22–24

Among trace ions present in human mineralized tissues, fluorine and zinc show a particularly promising behavior. Indeed, different from all the other substitutions (except for Sr at high concentrations), F⁻ substitution for OH⁻ causes an increase in the symmetry of the lattice, so that fluorine-substituted apatite shows higher stability and lower solubility than hydroxyapatite (HA). Notably, fluorine is expected to exert a positive influence against osteoporosis and to have antibacterial effect.⁴⁴ For these reasons, research on fluorine-substituted hydroxyapatite is attracting increasing attention for its use as bioactive and antibacterial material. Zinc also shows a high antibacterial effect if used at proper concentrations.^20,22

To pursue biomimetism and exploit the biological role of different ions, research is moving towards the use of multi-doped apatites.^{13,22,45–49} This is particularly important for infections, where different metal ions can inhibit different microbial populations, thus multi-doped apatites can be more effective than single-doped ones towards a larger number of bacterial strains.⁵⁰ However, obtaining multi-doped apatites is challenging, as each substitution causes distortion in the lattice, thus modifying the amount of other ions that can be entrapped. This becomes even more challenging when switching from bulk materials to coatings, where the capability of the technique to preserve the composition from the deposition target to the coating must also be taken into account. For this reason, the use of naturally derived biomaterials is being widely pursued and the deposition of coatings from biogenic sources to achieve multi-substituted coatings, and in particular that of bone, is raising increasing interest.^51–57

Deposition of targets from biogenic sources to obtain nanostructured and adhesive films has been explored by pulsed laser deposition, magnetron sputtering and pulsed electron deposition.^58–61 In this regard, the authors have demonstrated that nanostructured thin films having a composition perfectly resembling that of the deposition target can be achieved by ionized jet deposition (IJD). Thanks to their nanostructured surface and the high surface area, high ion release is obtained, permitting a sustained antimicrobial activity.^62–65 At the same time, the combination of biomimetic composition and nanostructured morphology permits mitigation of toxicity towards host cells and in some cases can promote differentiation towards an osteogenic lineage.^58,59,64

Together with the use of bone, biomaterials derived from seashells are also raising increasing interest. Seashells are a byproduct of the food industry, so their re-use for high added value applications is very promising in the view of circular economy and the use of sustainable sources for materials production. In addition, they have high availability and unique properties, including that of being multi-doped, that make them particularly suitable for application in biomaterials.⁶⁶ Calcium carbonate-based biomaterials have been proposed using unprocessed seashells, and conversion of carbonate in calcium phosphates has been largely explored, for the development of biomimetic materials, although nanostructured coatings deriving from seashells are yet to be explored.⁶⁶

Here, we propose the use of natural fluorapatite deriving from the shell of the brachiopod Lingula anatina as an antibacterial biomaterial, mimicking the composition of dentine and enamel, for orthopedics and dentistry. The L. anatina shell is an organic–inorganic composite material, where the inorganic phase is made of 4–8 nm crystals of carbonate-substituted fluorapatite in the form of francolite and comprises ca. 68% of the shell weight.^67,68 The organic fraction, which comprises the remaining 32% of the shell weight, is composed of proteins, glycosaminoglycans and chitin.^67,69,70 At the micron scale, the shell has a laminated structure made of alternating layers of predominantly organic and predominantly mineral phases. The L. anatina shell is of particular interest to orthopedics and dentistry, as its inorganic composition is close to that of bone and enamel, and it contains ion dopants (magnesium, zinc, sodium, manganese) that are important to promote bone regeneration but difficult to incorporate into synthetically produced hydroxyapatite.

The compositional features and antibacterial action of L. anatina are studied, for its use in the form of granulates, or nano/micro-powders, to be embedded in polymeric/ceramic scaffolds. Then, its deposition is investigated by IJD, to produce nanostructured biomimetic thin films.

The composition of the coatings is studied to investigate the capability of IJD to preserve the main phase and the substitutions that are present in L. anatina seashell. Then, nanostructured morphology is shown and antibacterial action of the films is verified against Gram-positive and Gram-negative bacterial strains, for application in orthopedics, dentistry and maxillofacial surgery.

2. Experimental

2.1. Materials

L. anatina dry seashells (unprocessed) were used for the tests. The sample was supplied by the Mostra Mondiale Malacologia (Cupra Marittima, AP, Italy) and it was collected in the Salu Sea (Philippines).

Films were deposited onto titanium–aluminum–vanadium microstructured disks and on glass slides, after cleaning in ethanol and water. Silicon wafers (Si) (p-type doped monocrystalline (100) native silicon, size 5 × 5 mm, thickness 1 mm, Fondazione Bruno Kessler, Trento, Italy) were also used as reference substrate for topographical characterizations. No surface preparation was carried out for any of the substrates, prior to deposition.

Synthetic fluorapatite was used for comparison sake, synthesized as described in ref. 71

2.2. Methods

2.2.1. Characterization of L. anatina seashell. Prior to its deposition, composition and morphology of L. anatina seashell were characterized.

Morphology was studied by Field Emission Gun Scanning Electron Microscopy (FEG-SEM, Tescan Mira3, CZ, working distance = 10 mm, voltage = 10 kV). FEG-SEM samples (fragments of lingula shell) were made conductive by sputtering with carbon before observation.

The mineralogical composition of L. anatina and its crystallinity were investigated by X-rays diffractometry (XRD, X’Pert PRO, Malvern Panalytical, Malvern, UK, 2θ = 5°–80°, scan step size 0.017°, time per step 50 s). Then, phase composition was also studied by Fourier transform infrared spectroscopy (FTIR-ATR, PerkinElmer Spectrum 2, acquisition parameters: resolution 1 cm⁻¹, accumulation 64 scans, data interval 0.5 cm⁻¹).

Both XRD and FT-IR were carried out on pulverized samples, obtained by manual grinding in agate mortar.

Elemental composition was tested by EDS and ICP (ICP-OES, Spectro Arcos-Ametek, Inductive Coupled Plasma Optical Emission Spectroscopy). For EDS, fragments of L. anatina shells were examined, without processing. The EDS analysis was performed using a Bruker probe coupled with the Tescan Mira 3 FEG-SEM. A working voltage of 10 kV was used and samples were made conductive by coating with carbon, as for the SEM observations. EDS spectra and maps were acquired to investigate distribution of calcium, phosphorous, zinc and fluorine in the shell. For ICP, after grinding, samples were digested in 4.5 mL ultrapure nitric acid and 0.5 mL hydrogen peroxide.

2.2.2. Antibacterial properties of lingula seashell. The antimicrobial activity of L. anatina was assessed against the two common human pathogens E. coli ATCC 8739 and S. aureus ATCC 6538P. The study was carried out by growing the bacterial cells in 50 mL tubes with 5 mL of Luria-Bertani (LB) liquid medium (NaCl 1%, tryptone 1%, yeast extract 0.5%) overnight at 37 °C under agitation at 130 rpm. For the tests, all the strains were diluted to reach an initial cellular concentration of 10⁵ CFU mL⁻¹. The assays were performed in 12-well microplates with 1 mL of LB as growth medium. Around a quarter of the lingula (around 1.2 cm²) was added to the cultures. The incubation was performed at 37 °C and 130 rpm. Bacterial cell enumeration was based on serial dilution method (using 20-μL volume from each culture) that was performed every hour in saline solution (NaCl 0.85%) and using LB agar plates as growth medium for colony forming units (CFU) count. Control experiments consisted in bacterial cultures grown on LB medium without the lingula addition. Each experiment was repeated in triplicate. All results are reported as mean ± standard deviation (SD) calculated from three replicates. Statistical analysis was performed using one-way Anova test. Differences were considered significant when p < 0.05.

The Minimal Inhibitory Concentration of both L. anatina shell and fluorapatite (used as a benchmark) was determined by culturing each strain in 5 mL LB in 6-well microplates supplied with shell powder and fluorapatite powder at different concentrations. After 24 hours of growth, serial dilutions and CFU counts were performed on LB agar plates. The minimal inhibitory concentration (MIC) was determined as the lowest concentration that prevented microbial growth.

2.2.3. Coatings deposition. Coatings were obtained by ionized jet deposition (IJD, Noivion Srl, Rovereto, Italy), as schematized in Fig. 1. During the process, the target (unprocessed L. anatina seashell) is ablated by a fast pulse (100 ns) of high energy (10 J) and high-density (109 W cm⁻²) electrons. Deposition parameters were selected based on (i) the uniformity of the plasma plume (possibility to obtain a plume at each pulse of the electron beam) and (ii) on preliminary data for deposition of calcium phosphates. As a consequence, the plasma plume was directed toward a substrate mounted at 8 cm distance from the target and a working voltage and an electron beam frequency of 17 kV and 7 Hz, respectively, were selected. Deposition time was set to 20 minutes, as it is the highest duration that can be achieved by using one target.

Fig. 1 IJD deposition scheme.

The deposition is carried out in vacuum (the chamber is initially evacuated down to a base pressure of 1.0 × 10⁻⁷ mbar by a turbomolecular pump – EXT255H, Edwards, Crawley, England), then pressure is raised by a controlled flow of oxygen up to 3 × 10⁻⁴ mbar. A L. anatina seashell was used as supplied, after washing in deionized water and ethanol and drying to constant weight. No processing of the shell was carried out to remove the organic components prior to deposition. For the deposition, one half of the shell was mounted on the target holder by using adhesive tape and used as a target.

2.2.3.1. Coatings morphology and composition. After deposition, morphology and composition of the coatings were studies to assess compliance to the objectives, i.e., obtaining nanostructured and biomimetic thin films, having a composition as close as possible to that of the L. anatina seashell.

Coatings morphology was studied after deposition on titanium alloy disks, by FEG-SEM, as described above for the target. Based on FEG-SEM images at 50 000× magnification, the maximum and minimum dimension of the aggregates that constitute the films was measured by ImageJ software (National Institutes of Health, USA). To analyze the distribution in aggregates diameters, 3 non overlapping areas of two samples were selected, each composed by at least 45 clearly distinguishable aggregates. All areas had the same dimensions and were chosen within images at a 50 000× magnification.

Composition was investigated by FT-IR, EDS and ICP, as described above for the shell before deposition. Film composition by ICP and FT-IR is measured on silicon wafers.

Then on the coting 2D-GIWAXS images were collected at the XRD1 beamline of the ELETTRA Synchrotron radiation facility (Trieste, Italy), by using an incident X-ray beam of 1A and 200 × 200 μm² size and a 2D Pilatus detector placed at 90 cm from the sample position. The incident angle was fixed at α_i = 0.05 deg, 0.1 deg and 0.15 deg, to investigate different thickness of the samples, and moved laterally to probe different area of the sample.

2.2.3.2. Substrate effect on coatings properties. Coatings cross-section was also observed by FIB to study film thickness, morphology and presence/absence of pores and/or defects in the cross section. For FIB, coatings were deposited onto different materials, to study the impact of the substrate characteristics on coatings thickness and morphology. FIB milling and SEM imaging were done using a Zeiss Crossbeam 550 equipped with an inlens, secondary electrons, backscattered electrons detectors and an Oxford instruments energy-dispersive X-ray spectroscopy detector (EDS). Milling was done at an ion beam accelerating voltage of 30 kV and beam current of 7 nAmp, while SEM imaging was performed with an accelerating voltage of 5 kV using the inlens and backscattered electrons detector.

To assess the stability of the coatings, dissolution tests were performed in complete culture medium (sterile alpha-MEM, Minimum Essential Medium Eagle, Sigma Aldrich, supplemented with 1 wt% 100 U mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin to avoid bacterial contamination and 10% fetal bovine serum). Samples (2 for each timepoint) were submerged in medium at pH 7.4 for 24 h, 7 days, 14 days and 21 days, respectively. The desired pH was obtained by addition of NaHCO₃ (2.2 g l⁻¹). Films were placed in 24 wells and 1 ml medium was put into each well. Wells plates were sealed and kept in a humidified incubator at 37 °C, 95% air and 5% CO₂. Medium was refreshed every 72 h.

At each timepoint, stability was evaluated by observing samples morphology by SEM, performed as described above. SEM/EDS was performed on at least 2 non-overlapping areas of each sample, 1 close to the center and 1 on the samples edge.

The antimicrobial activity of deposited L. anatina seashell coatings was assessed by growing the microorganisms in 50 mL tubes with 5 mL of Luria-Bertani (LB) liquid medium (NaCl 1%, tryptone 1%, yeast extract 0.5%) overnight at 37 °C under agitation at 130 rpm. For the tests, all the strains were diluted to reach 10⁵ CFU mL⁻¹. The assays were performed in 48-well microplates with 400 μL of LB medium. A glass piece 0.5 × 0.5 cm with deposited lingula shield on it was added to the cultures. The incubation was performed at 37 °C and 130 rpm. Serial dilutions of 20-μL of cultures were performed every hour in saline solution (NaCl 0.85%) and spread onto LB agar plates for CFU counting. Control experiments consisted in bacterial cultures with LB medium only. Each experiment was repeated in triplicate.

Biocompatibility of the coatings on glass slides was evaluated by measuring the cell viability of MG-63 osteoblast-like cells after the exposure to the coatings. For the test, 10 × 10³ MG-63 were seeded at the bottom of a 48-well plate and the samples, previously sterilized under UV ray for 2 hours, were added at the well and placed in direct contact with cell monolayer, and maintained in culture for 7 days. Uncoated glass slides were used as control. Cell viability was monitored by Alamar Blue assay at 1, 3 and 7 days. Briefly, to perform Alamar Blue assay, the culture medium was removed, replaced with the Alamar Blue solution prepared as 10% v/v in fresh culture medium, then incubated at 37 °C, with 95% humidity for 3 h. The fluorescence was quantified in a microplate spectrophotometer (Infinite F200 PRO, TECAN, Mannedorf, Switzerland) at 535 nm excitation and 590 nm emission wavelengths. Data expressed as Relative Fluorescence Units (RFU), are reported as mean ± standard error of triplicates.

To exclude any potential toxic effect of the material's degradation by-products, the conditioned medium (CM), obtained from keeping the samples in culture medium for 24 h, was added to previously seeded cell monolayer of MG-63 and maintained in culture for further 24 hours (37 °C, 95% humidity). The CM was diluted 100 : 0, 50 : 50, 25 : 75, and 5 : 95 with fresh culture medium. Cells cultured without administration of the conditioned medium were used as control.

3. Results & discussion

3.1. Characterization and antimicrobial efficacy of lingula shell

XRD spectra of L. anatina seashell are reported in Fig. 2. All peaks correspond to fluorapatite (JCPDS 15-0876), with a high crystallinity.⁷² EDS data (Table 1 and Fig. S2, ESI†) show the presence of fluorine at an approximate concentration of 0.06 wt%, a calcium to phosphate ratio of 1.75 and the presence of Mg and Na ions. Elemental composition, as assessed by ICP (Table 2), shows that, together with fluorine, many elements are present in the shell, including magnesium, sodium, potassium, silicon, strontium and zinc, all being present in bone and enamel and hence increasing biomimicry.⁹

Fig. 2 (a) EDS and (b) XRD performed on L. anatina seashell.

Table 1 EDS data for L. anatina seashell and the lingula-derived coatings

	F [wt%]	Ca/P at% ratio
Shell	0.06	1.75
Coating	6.33	1.60

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Table 2 L. anatina seashell and coating composition, as assessed by ICP-MS

	Ca	K	Mg	Mn	Na	P	Si	Sr	Zn
	ppm	ppm	ppm	ppm	ppm	ppm	ppm	ppm	ppm
	Average ± st dev
Shell	1299.00 ± 0.04	70.40 ± 2.65	95.11 ± 0.01	0.31 ± 0.01	246.00 ± 0.03	704.00 ± 0.00	133.40 ± 0.17	3.307 ± 0.00	0.98 ± 0.01
Coating	4.48 ± 0.29	0.13 ± 0.01	0.15 ± 0.00	0.01 ± 0.00	0.19 ± 0.02	1.40 ± 0.08	0.01 ± 0.00	0.03 ± 0.00	0.165 ± 0.01

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FT-IR spectra (Fig. 3) indicate a very strong band at 1025 cm⁻¹, with a shoulder at 1092 (ν₃ antisymmetric PO stretching mode), and bands at 602 cm⁻¹ and 560 cm⁻¹ (ν₄ PO₄ antisymmetric bend), all compatible with natural fluorapatite.^73–75 The presence of carbonates is detected by the bands at 1450, 1419 (asymmetrical and symmetrical stretching modes of ν₃CO3) and 870 (ν₂CO₃, characteristic of beta-type substitution in HA^73–77) cm⁻¹, indicating carbonate-substitution in fluorapatite. Finally, the organic phase (chitin) is revealed by the bands at 1640, 1538 and 1231 cm⁻¹, to be ascribed to amide II (N–H bending) and vC–O absorption, respectively.⁷⁸

Fig. 3 FT-IR of L. anatina seashell before and after deposition in the form of nanostructured coatings.

SEM and EDS data (ESI,† Fig. S1 and S2), show that the outer surface of the shell is composed by a uniform fluorapatite layer.

For both strains, the MIC of the L. anatina seashell is lower compared to the MIC of the fluorapatite. In particular, the L. anatina MIC against E. coli is 80 mg mL⁻¹ and three times lower than fluorapatite, whereas the MIC against S. aureus is 20 mg mL⁻¹ and two times lower than fluorapatite ref. 79 and 80. These results indicate higher antibacterial efficacy compared to the synthetic analog not including the organic part. The increased antibacterial activity of the L. anatina seashell is likely due to the combination of the two antibacterial ions composing the inorganic part (about 68% in weight), mainly fluorine and zinc, together with the organic part composed of chitin/chitosan (about 32% in weight).

To study the antibacterial effect of the mineral part (i.e. the one that can be deposited in coating) and the effect of the organic part, we deproteinized the shell by heating it at 400 °C (ESI,† Fig. S4 and Table S1). Indeed, past results on Discinisca tenuis that has the same inorganic and organic composition as lingula, but a different structural organization, indicate that no modifications occurred at temperatures <700 °C, except for the decomposition of the organic part.⁸¹ In our work, the results show that while the efficacy against S. aureus is not significantly modified after removing the organic part (99.37% CFU reduction), the antibacterial efficacy against E. coli drops significantly (84.15% CFU reduction, Fig. S4 and Table S1, ESI†). This result can be related with the mechanism of action of chitin/chitosan that seems to be active against the negatively charged lipopolysaccharides (LPS) and proteins located on the bacterial cell surface leading to the disruption of the cell envelope structure.⁸² Therefore, the chitin/chitosan part of the L. anatina seashell significantly contributes to the antibacterial activity exerted against E. coli that is a Gram negative strain having an outer membrane with LPS molecules.⁸³ Further, E. coli was more susceptible to fluorapatite as compared to L. anatina seashell (MIC of fluorapatite is higher than L. anatina seashell in Table 3), indicating that the organic part of the shell (composed by chitin/chitosan) greatly contributes to the antibacterial effect against Gram negative strains. On the other hand, the activity against S. aureus is not significantly modified by the absence of chitosan suggesting that the inorganic part of the L. anatina seashell (fluorine and zinc) plays a critical role in the antibacterial activity towards this Gram positive strain. This is in line with previous studies revealing a significant inhibition exerted by zinc-based nanocoatings against S. aureus under both planktonic and biofilm growth conditions.⁸⁴ The antibacterial activity of these coatings is stronger against S. aureus compared to E. coli, probably due to an improved binding of zinc onto S. aureus cell surface or other events related with zinc cellular intake and interference with cellular mechanisms causing damage events including cell wall disruption, reactive oxygen species (ROS) production, and ATP synthesis inhibition.^62,84–86

Table 3 Minimal inhibitory concentration of L. anatina seashell compared to fluorapatite against the growth of Escherichia coli and Staphylococcus aureus strains

	Fluorapatite (mg mL^−1)	L. anatina seashell (mg mL^−1)
Escherichia coli	240	80
Staphylococcus aureus	40	20

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3.2. Nanostructured coatings deposition and characterization

Ionized jet deposition (IJT) was used to obtain nanostructured coatings of fluorapatite from the L. anatina seashell on a silica wafer substrate. The obtained coating was composed of aggregates with sizes ranging from 100 nm up to 1 μm, in many cases made of particles 20 nm in diameter (Fig. 4), characteristic of the deposition of CaP-based and ceramic-like materials by IJD. The surface of samples was homogeneous and no defects were noticed in any part of the samples.

Fig. 4 Morphology of the nanostructured films obtained by IJD deposition of L. anatina seashell. Increasing magnification is shown from (a) to (f).

In FT-IR spectra (Fig. 3), bands observed for the coatings are the same as for the mineral part of L. anatina and are compatible with fluorapatite and carbonated hydroxyapatite. In our case, a distinction between fluorapatite and hydroxyapatite by FT-IR is not possible, due to the low crystallinity of the coatings, making FT-IR bands broad and ill-defined, while bands characteristic of carbonate substitutions can be clearly identified. The bands corresponding to the organic components are absent, showing their degradation during the IJD process. No other bands are assessed, so no metastable or decomposition phases are present in the film, as reported for similar techniques.

Regardless of the sample position and the incident angles the 2D-GIWAXS images show no diffraction signals but only diffused rings, typical of amorphous materials (Fig. 5(a)). The diffraction patterns obtained by integrating the intensity all over the image, indicate broad large peaks corresponding to periodicities of 1 Å, 1.8 Å and 3.14 Å (Fig. 5(b)).

Fig. 5 (a) 2D-GIWAXS image of coated film recorded by using incident angle = 0.1°, (b) corresponding integrated intensity diffraction profile.

This indicates that samples obtained by IJD are highly amorphous, consistently with what observed for biogenic hydroxyapatite.⁶¹

EDS data show that the Ca/P ratio is similar to that of the target material. In addition, fluorine is transferred in the coating, in a percentage higher than that of the target, indicating a preferential sputtering of fluorine (Table 1). ICP-MS data show that all trace elements present in the coating, including antibacterial zinc, are preserved in the coating, making it fully biomimetic (Table 2), although some preferential sputtering is observed. Indeed, starting from data of ICP-MS, we calculated the ratio between each substituent and calcium (Table S2, ESI†). Data indicate that all elements are transferred from the target to the coating, with K, Mg and Na being present in a similar, though lower concentration in the coating compared to the target, while Mn, Sr and, mostly, zinc, showing a preferential sputtering compared to calcium.

Altogether, however, our data show an excellent transfer in the composition between the target material and the coating with both the main mineral phase and all dopants present in the shell preserved in the coating.

Interestingly, from the point of view of IJD performance, if the seashell is not appropriately washed, also the NaCl crystals present in the seashell are decomposed and reconstructed in the coating, without changes of phase (Fig. S5, ESI†). This, of course, is detrimental for application in biomaterials, but is very important in terms of deposition performance, as it demonstrates the possibility to keep different phases in the target to be separated also in the coating, even when both phases are complex (here, a multi-doped hydroxyapatite and sodium chloride are present), have high tendency to incorporate foreign ions (apatite) and/or when the two phases share the same ions (sodium).

3.3. Substrate influence on coatings characteristics

To study the properties of the coatings depending on the substrate and their applicability to different materials, we carried out a Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM, Fig. 6–8) study comparing deposition on titanium alloy disks and on silicon wafers as reference materials. We then assessed the substrate influence on film thickness and microstructure.

Fig. 6 FIB analyses of lingula films on glass substrates: imaging by secondary electron detector at lower (a) and higher (c) magnification; backscattered electrons imaging at (b) lower and (d) higher magnification; (e) EDS maps.

Irrespective of the substrate, all films have a thickness of about 650 nm and a nanostructured surface morphology. However, significant modifications are observed when changing the substrate, as deposition onto polished titanium results in a rougher coating, characterized by the presence of coarser aggregates, while the films on glass and silicon are smoother.

When the coating was deposited onto glass and silicon, we observed the deposition of a homogeneous calcium phosphate film, with no evidence of porosity or defects. Some isolated coarser aggregates appeared on the surface.

While films on glass and silicon wafer were smooth (Fig. 6 and 7), the ones on Ti substrate were rough and non-uniform (Fig. 8). Although titanium disks were polished, we cannot exclude an influence of the substrate surface roughness on this parameter. As for the other coatings, no obvious internal structure or porosity is observed in the films.

Fig. 7 FIB analyses of lingula films on silica wafers: (a) imaging by inlens detector at lower (a) and higher (c) magnification; backscattered electrons imaging at (b) lower and (d) higher magnification; (e) EDS maps.

Fig. 8 FIB analyses of lingula films on microstructured titanium–aluminum–vanadium alloy substrates: (a) imaging by inlens detector at lower (a) and higher (c) magnification; backscattered electrons imaging at (b) lower and (d) higher magnification; (e) EDS maps.

These data indicate that some of the characteristics of the coatings, such as morphology, significantly depend on the substrate composition, suggesting that a specific optimization is needed for each substrate to be coated. On the other hand, other characteristics, such as thickness, are not influenced significantly by the substrate, while they strongly vary depending on the target.^87,88 Interestingly, the extent of this dependence and the characteristics that are influenced is determined by the class of materials to which they are applied. Indeed, in the case of deposition onto polymers,^58,65 we observed that thickness significantly varies depending on the polymer surface charge, and so do coating composition, while surface morphology remains essentially unaltered.

3.4 L. anatina seashell coatings performance

Coatings are biocompatible, as reported in Fig. 9(a) where osteoblast-like cells MG-63 in direct contact with coating samples reported a good cell viability when compared to CTRL (no coating), with a notable cell proliferation over time and until 7 days of culture (Fig. 9(a)). When CM derived from coating was added to MG-63 cells, no cytotoxic effects due to the by-products are detected after 1 day of maintenance, except for a slight effect for the highest concentrated dilution (Fig. 9(b)).

Fig. 9 (a) Cell viability of MG-63 with and without coating sample after 1 day (T1-1d), 3 days (T2-3d), and 7 days (T3-7d) of culture. (b) Cell viability of MG-63 after 24 h of maintenance with CM derived from coating at different dilutions with fresh culture medium (100 : 0, 50 : 50, 25 : 75, and 5 : 95). In both (a) and (b) panels, the dashed line is for CTRL at just seeded.

Stability tests (Fig. 10) show that the coatings are stable in alpha-MEM medium until 21 days, so a prolonged antimicrobial effect is to be expected (data in progress). This is in contrast to coatings obtained by IJD of stoichiometric and ion-doped apatite in the absence of fluorine, which completely dissolve within 7 days.⁶¹ We postulate that this enhanced stability of the coatings made from the L. anatina seashell derives from the fluorine content, which is known to reduce the solubility of apatite. The higher stability of these films can be especially promising for thermo-sensitive substrates, for which deposition in temperature is not permitted. When dissolution occurs, after 21 days, it happens without the formation of cracks or detachments, which would be detrimental, as they could cause inflammation and toxicity in vivo. In addition, dissolution of the coating is homogeneous, and we did not observe faster dissolution in specific areas of the coatings, which might suggest the presence of different phases in the coating. As a consequence, based on XRD, FT-IR and EDS data, we believe that the coating is composed by multi-substituted hydroxyapatite containing fluorine and carbonates (alongside the other mentioned ions) and not by a mix of fluorapatite and hydroxyapatite.

Fig. 10 Stability of L. anatina seashell coatings after immersion in medium for (a) and (b) 6 hours, (c) and (d), 14 days, (e) and (f) 21 days, as assessed by FEG-SEM.

Coatings have antibacterial efficacy against both E. coli and S. aureus strains (Fig. S6, ESI†), with inhibition percentages of 88.83% and 99% of CFUs number compared to the control experiments, respectively. This correlates with the higher antibacterial efficacy of the seashell (fluorine and zinc-based), towards Gram-positive bacterial strains. On the other hand, the percentage of growth inhibition for the two bacteria is similar to that observed testing the deproteinized shell (Fig. S4, ESI†).

4. Conclusions

For the first time, we explore the possible use seashells and, in particular, of L. anatina seashell as a biomaterial for orthopedic and dental applications. Results obtained show that L. anatina seashell is composed by a carbonated fluorine-doped apatite, also containing traces of zinc, magnesium, strontium and manganese. All these trace ions are contained in bone and dental enamel, thus making the coatings obtained from L. anatina biomimetic. Among the trace ions, fluorine and zinc also show potential antimicrobial activity. Furthermore, for the first time, we demonstrate that, thanks to the combination of this unique mineral phase and an organic part constituted by chitin, L. anatina seashell has high antibacterial activity against both Gram-positive and Gram-negative strains, thus resulting more antibacterial than synthetic fluorapatite, as denoted by the significantly lower Minimal Inhibitory Concentration (MIC) of L. anatina seashell compared to fluorapatite. The mineral part alone shows higher efficacy against the Gram positive S. aureus strain and a more moderate effect on the Gram negative E. coli strain.

Upon deposition by IJD, nanostructured biomimetic coatings can be obtained, having the same composition as the inorganic phase of L. anatina seashell. Indeed, we show that IJD permits a good conservation of the target stoichiometry, avoiding the formation of secondary products and/or a poor conservation of ion doping, which often occur with many plasma-assisted techniques.

The thickness of the coatings (650 nm) is independent of the substrate to which it is applied, while surface morphology strongly depends on the characteristics of the device. Coatings are biocompatible and stable for over 21 days of immersion in α-MEM medium. In addition, they cause a high reduction of viability on S. aureus, and a moderate effect on E. coli. The effect is dose-dependent, thus causing the delay/impairment of bacterial growth when supplied at low concentration and exerting a toxic/killing effect towards bacteria when added at high concentration.

Our results show that deposition from seashells is possible by IJD, and represent a promising route to obtain biomimetic biomaterials. In addition, thanks to its biomimicry and special properties, we believe lingula shell is a very promising material for orthopedics and dentistry, to produce nanostructured coatings and impart antibacterial functionalization to different devices.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by funding from the Royal Society of Edinburgh Saltire International Collaboration Award, grant no. 1887, to FN, GF and GG. NB acknowledges funding from 5 × 1000 Anno 2019, Redditi 2018 “MEDICINA RIGENERATIVA E RIPARATIVA PERSONALIZZATA PER LE PATOLOGIE DEI TESSUTI MUSCOLO-SCHELETRICI E LA CHIRURGIA RICOSTRUTTIVA ORTOPEDICA”. FIB-SEM was performed at the facility at the University of Edinburgh (EPSRC grant no. EP/P030564/1). Dr Michele Iafisco (National Research Council, Istituto di Scienza, Tecnologia e Sostenibilità per lo Sviluppo dei Materiali Ceramici, ISSMC-CNR, Faenza, Italy) is kindly acknowledged for providing synthetic fluorapatite.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3tb02454g

‡ Present address: Department of Chemistry, Materials and Chemical Engineering “G. Natta”, Politecnico di Milano, Milan, Italy.

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