Use of the Sol–Gel Method for the Preparation of Coatings of Titanium Substrates with Hydroxyapatite for Biomedical Application

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Article Use of the Sol–Gel Method for the Preparation of Coatings of Titanium Substrates with Hydroxyapatite for Biomedical Application

Michelina Catauro 1,* , Federico Barrino 1 , Ignazio Blanco 2 , Simona Piccolella 3 and Severina Paciﬁco 3 1 Department of Engineering, University of Campania “Luigi Vanvitelli”, Via Roma 29, I-813031 Aversa, Italy; [email protected] 2 Department of Civil Engineering and Architecture and UdR-Catania Consorzio INSTM, University of Catania, Viale Andrea Doria 6, 95125 Catania, Italy; [email protected] 3 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, Via Vivaldi 43, 81100 Caserta, Italy; [email protected] (S.P.); severina.paciﬁ[email protected] (S.P.) * Correspondence: [email protected]

Received: 27 December 2019; Accepted: 24 February 2020; Published: 26 February 2020

Abstract: Hydroxyapatite (HA) was coated onto the surface of commercially pure titanium grade 4 (a material generally used for implant application) by a dip coating method using HA sol. Hydroxyapatite sol was synthesized via sol–gel using Ca(NO ) 4H O and P O as precursors. The surface of the HA 3 2· 2 2 5 coating was homogeneous, as determined by scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared (ATR-FTIR), and X-ray diffraction (XRD), which allowed the materials to be characterized. The bioactivity of the synthesized materials and their efficiency for use as future bone implants was confirmed by observing the formation of a layer of hydroxyapatite on the surface of the samples soaked in a fluid simulating the composition of human blood plasma. To verify the biocompatibility of the obtained biomaterial, fibroblasts were grown on a glass surface and were tested for viability after 24 h. The results of the WST-8 analysis suggest that the HA systems, prepared by the sol–gel method, are most suitable for modifying the surface of titanium implants and improving their biocompatibility.

Keywords: sol–gel synthesis; hydroxyapatite; titanium substrate coating; biocompatibility

1. Introduction The aim of tissue engineering is to develop substitutes, organic or synthetic, which are able to restore or maintain the function of damaged tissue within the human body. This science is the synergistic union between life sciences and engineering [1]. Tissue engineering should ideally produce substitute tissue that can “grow” with the patient, overcoming the limitations of conventional treatments that are based on biomaterial implant and organ transplantation [2]. Scaffolds must meet requirements, such as biocompatibility, surface topography, and chemistry, in order to promote cell adhesion, proliferation and differentiation, controlled biodegradability, and bioactivity [3]. Taking into account these fundamental characteristics, different types of materials (natural, synthetic, semisynthetic, and hybrids) have been synthesized to develop scaffolds according to the specific tissue that requires regeneration, with focused attention on the concept of controlled release of biological molecules or cell differentiation induction [4,5]. Scaffolds for bone tissue engineering must have suitable mechanical properties. For this reason, ceramic materials such as hydroxyapatite, silica, zirconia, and tricalcium phosphate have been considered. These materials have proven to be

Coatings 2020, 10, 203; doi:10.3390/coatings10030203 www.mdpi.com/journal/coatings Coatings 2020, 10, 203 2 of 8

Coatings 2019, 9 FOR PEER REVIEW 7 biocompatible and bioactive, which are basic characteristics necessary in the orthopedic or dental fields [6,7Hydroxyapatite]. (HA) has attracted much attention as a material for artificial bones, scaffolds for tissueHydroxyapatite engineering (HA), and has chromatographic attracted much packaging attention as [8 a]. materialDue to the for chemical artificial bones,similarity sca ffbetweenolds for HA tissueand engineering, the mineralized and chromatographic bone of human tissue, packaging synthetic [8]. DueHA toexhibits the chemical strong affinity similarity to host between hard HA tissues andand the mineralizedis able to form bone chemical of human bonds tissue, with synthetic the host HAtissue. exhibits Metallic strong implants, affinity on to the host other hard hand, tissues do not andinteract is able to with form the chemical biological bonds environment with the host. H tissue.owever, Metallic metallic implants, alloy is on a bioinert the other material hand, do [9,10 not]. In interactrecent with years the, biological coatings that environment. can be applied However, to metal metallic alloys alloy have is been a bioinert developed material. These [9,10 have]. In recentbeen used years,in coatings the biomedical that can be field applied to improve to metal interaction alloys have with been hard developed. tissue These[11,12] have. Nanocrystalline been used in the HA is biomedicalsynthesized field toby improve various interactionprocesses, withsuch hardas hydrothermal tissue [11,12]. [ Nanocrystalline13], mechanochemical HA is synthesized[14], precipitation by various[15] processes,, hydrolysis such [16 as], hydrothermaland sol–gel methods [13], mechanochemical [17]. The sol– [gel14], methodprecipitation is able [15 ], to hydrolysis improve [ the16], HA andchemical sol–gel methods homogeneity [17]. The in comparison sol–gel method with is other able to techniques improve, the because HA chemical it involves homogeneity a mixing in at the comparisonmolecular with level other of techniques, calcium and because phosphorus it involves precursors. a mixing T athis the technique molecular can level also of calciumform thin and film phosphoruscoatings precursors.with a simple This process technique such can as dip also coating. form thin film coatings with a simple process such as dip coating.The dip coating technique allows substrates of different materials to be coated with complex shapesThe dip [18 coating–23]. technique allows substrates of different materials to be coated with complex shapes [18In– 23the]. present work, synthetic HA was synthesized by using the sol–gel method. Hydroxyapatite solIn thewas present used to work, coat metallic synthetic substrate HA was synthesizedtitanium grade by using4 (Ti-4), the which sol–gel is method.widely used Hydroxyapatite in implants for sol wasorthopedic, used to coatdental metallic, and orthodontic substrate titanium wires. The grade dip 4 coating (Ti-4), whichtechnique is widely was used used to in make implants a thin for layer orthopedic,on substrates. dental, Scanning and orthodontic electron wires. microscope The dip coating(SEM), attenuated technique was total used reflectance to make aFourier thin layer transform on substrates.infrared Scanning (ATR-FTIR) electron, and microscope X-ray diffraction (SEM), attenuated(XRD) were total then reflectance used for Fourier chemical transform and morphological infrared (ATR-FTIR),coating characterizations. and X-ray diffraction The (XRD) objective were of then the usedcoatings for chemicalis to combine and morphological the favorable coating mechanical characterizations.properties of titanium The objective with the of the excellent coatings biological is to combine properties the favorable of HA. For mechanical that reason, properties after coating, of titaniumbioactivity with the and excellent biocompatibility biological of properties the coated of substrates HA. For that were reason, preliminarily after coating, evaluated bioactivity and compared and biocompatibilitywith those of ofuncoated the coated substrates. substrates Bioactivity were preliminarily tests were performed evaluated andby soaking compared samples with in those simulated of uncoatedbody substrates. fluids (SBF) Bioactivity [24]. Biocompatible tests were performedproperties wereby soaking assessed samples through insimulated WST-8 tests body on fluidsNIH-3T3 (SBF)murine [24]. Biocompatible fibroblasts. properties were assessed through WST-8 tests on NIH-3T3 murine fibroblasts.

2. Materials and Methods 2. Materials and Methods 2.1. Sol–Gel Synthesis 2.1. Sol–Gel Synthesis Hydroxyapatite for coating was synthesized using two diﬀerent chemical reagents as precursors. Hydroxyapatite for coating was synthesized using two different chemical reagents as Phosphorous pentoxide (0.006 mol) (P2O5, Sigma Aldrich, St. Louis, MI, USA) and calcium nitrate precursors. Phosphorous pentoxide (0.006 mol) (P2O5, Sigma Aldrich, St. Louis, MI, USA) and calcium tetrahydrate (0.02 mol) (Ca(NO3)2 4H2O, Sigma Aldrich) were dissolved in absolute ethanol (EtOH, nitrate tetrahydrate (0.02 mol) ·(Ca(NO3)2 ∙ 4H2O, Sigma Aldrich) were dissolved in absolute ethanol 99.8% Sigma-Aldrich) to form 1 M and 2.5 M solutions respectively. NH4OH was utilized to adjust (EtOH, 99.8% Sigma-Aldrich) to form 1 M and 2.5 M solutions respectively. NH4OH was utilized to the pH to 11. The molar ratio of precursors Ca/P was 1.67, which is the desired ratio observed in adjust the pH to 11. The molar ratio of precursors Ca/P was 1.67, which is the desired ratio observed hydroxyapatite. The ﬂow chart of the synthesis is depicted in Figure1.AP 2O5 and ethanol solution in hydroxyapatite. The flow chart of the synthesis is depicted in Figure 1. A P2O5 and ethanol solution was stirred for 24 h to obtain a clear solution that was added dropwise to the stirred calcium nitrate was stirred for 24 h to obtain a clear solution that was added dropwise to the stirred calcium nitrate solution. Afterwards, the solution was stirred slowly for 2 h and left at room temperature, protected solution. Afterwards, the solution was stirred slowly for 2 h and left at room temperature, protected from dust for 24 h. from dust for 24 h.

FigureFigure 1. Schematic 1. Schematic ﬂow flow process process chart chart for the for synthesis the synthesis of hydroxyapatite of hydroxyapatite (HA). (HA).

2.2. Coating Procedure

Hydroxyapatite sol was used to coat commercially pure titanium grade 4 disks with 8 mm diameter (Sweden & Martina, Padua, Italy). A KSV LM dip coater was used for depositing sol–gel

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2.2. Coating Procedure Hydroxyapatite sol was used to coat commercially pure titanium grade 4 disks with 8 mm diameter (Sweden & Martina, Padua, Italy). A KSV LM dip coater was used for depositing sol–gel coatings. Before coating, the substrates were washed with acetone and then subjected to a passivation process using HNO3 65 wt%. The coating procedure was carried out 24 h after synthesis, and a withdrawal speed equal to 15 mm/min was used. The coated substrates were heated at 600 and 1000 ◦C for 2 h.

2.3. Chemical and Morphological Characterization

The chemical nature of the hybrid materials (600 ◦C untreated and 1000 ◦C treated) was investigated by X-ray diffraction analysis using a Philips diffractometer (Philips Electronic Instruments Co., Model PW 1730, Eindoven, Netherlands). Powder samples were scanned from 2Θ = 10 to 80◦ using CuKα radiation. Both heated and non-heat-treated HA samples were characterized chemically by ATR-FTIR and recorded on a Prestige-21 FTIR spectrometer equipped with an AIM-8800 infrared microscope (Shimazu, Tokyo, Japan), using the incorporated 3 mm diameter Ge ATR semicircular prism. Spectra 1 1 were acquired in the 4000–650 cm− range, using 45 scans and 8 cm− resolution. Spectra analyses were completed using Prestige software (IRsolution, version 1.10). Coating morphologies were investigated by a scanning electron microscope (SEM) Quanta 200 FEI (Europe Company, Eindhoven, Netherlands) fitted with energy dispersive X-ray spectroscopy (EDS). Samples were fixed on an aluminum stub with colloidal graphite and gold metalized using a K550X Sputter Coater (Emitech, Ashford, UK).

2.4. In Vitro Bioactivity The potential bone-bonding ability of a material when implanted in vivo can be predicted by the apatite-forming-ability test in vitro, in accordance with Kokubo et al. [24]. The test was carried out by soaking the titanium substrates, uncoated and coated, with HA after heating at 600 and 1000 ◦C for 7, 14, and 21 days, in a simulated body ﬂuid (SBF). This is a solution prepared by adding several salts to distilled water in order to give a concentration of ions similar to those of human blood plasma. As the ratio between the total surface area exposed to the solution and its volume aﬀects the formation of the HA layer, it was kept equal to 10. A water bath was used to maintain the polystyrene bottles, where the samples in the solution were placed at 37 ◦C. In order to avoid the depletion of ionic species in the SBF solution due to biomineral nucleation, the solution was replaced every 2 days. SEM/EDX analysis was performed after each soaking time in order to evaluate the formation of a hydroxyapatite layer on the surface of the air-dried samples.

2.5. WST-8 Assay To evaluate the coatings’ biocompatibility, NIH-3T3 murine fibroblasts (ATCC, Manassas, Virginia, USA) were seeded on Ti-4 coated and uncoated substrates. Cell viability was tested via WST-8 assay (coated and uncoated substrates. Cell viability was tested via WST-8 assay WST-8 [2-(2-methoxy-4-nitrophenyl) -3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt], which is water-soluble and purple in color, can be converted by mitochondrial dehydrogenases in living cells into yellow formazan soluble in culture medium. Thus, the formazan amount is directly proportional to the living cells number. NIH-3T3 cells were grown in a humified chamber at 37 ◦C, with 5% CO2. DMEM medium (Gibco, Carlsbad, CA, USA) was supplemented with 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin. NIH-3T3 cells were seeded on the surface of coated and uncoated Ti-4 disks, previously placed on the bottom of a 24-well plate. Cell density was equal to 5 103. After 24 h exposure time, a WST-8 viability test was carried out. The absorbance was measured × at 450 nm using a Victor3™ multilabel plate reader (PerkinElmer, Shelton, CT, USA). Cells grown on uncoated Ti-4 disks were defined as having 100% vitality. Cells grown on Ti-4 coated and uncoated substrates were observed by an inverted phase contrast and brightfield Zeiss Primo Vert Microscope (Jena, Germany), and representative images were acquired without specific staining. CoatingsCoatings 20192020, 9 ,FOR10, 203 PEER REVIEW 47 of 8

3. Results 3. Results 3.1. Spectroscopic and Structural Characterization 3.1. Spectroscopic and Structural Characterization The XRD results of sol–gel untreated (a), treated at 600 °C (b), and treated at 1000 °C (c) are The XRD results of sol–gel untreated (a), treated at 600 ◦C (b), and treated at 1000 ◦C (c) are shown shown in Figure 2. The sintering temperature plays an important role in HA formation. In fact, in Figure2. The sintering temperature plays an important role in HA formation. In fact, increasing the increasing the sintering temperature causes the samples to show several distinct and narrow peaks, sintering temperature causes the samples to show several distinct and narrow peaks, which suggests which suggests that there is an increase in crystallite size. that there is an increase in crystallite size.

FigureFigure 2. 2.X-rayX-ray diffraction diﬀraction (XRD) (XRD) results results of ofHA HA sol sol–gel–gel (a) (auntreated) untreated,, (b) ( btreated) treated at at600 600 °C◦, C,and and (c) (c) treatedtreated at at1000 1000 °C.◦ C.

InIn spectra spectra of ofheat heat-treated-treated samples samples,, typical typical peak peak positions positions correspond correspondinging to toHA HA reflections reflections were were observedobserved,, including including those those with with higher higher intensity asas wellwell as as the the peak peak positions positions (2Φ (2Ф)) at 27.76,at 27.76, 31.80, 31.80, 32.26, 32.26,33, and33, and 53.06, 53.06 which, which were were in line in withline with the (002), the (002) (211),, (211), (112), (112), (300), (300), and (004)and (004) reflections reflections of HA of [ 25HA]. [25]. ATR-FTIR spectra of the hydroxyapatite coating untreated (a), treated at 600 ◦C (b), and treated at 1000ATR◦C- (c)FTIR are spectra reported of the in Figure hydroxyapatite3. coating untreated (a), treated at 600 °C (b), and treated at 1000In °C spectra (c) are ofreported untreated in Figure samples, 3. strong signals due to the asymmetric and symmetric stretching 1 1 of nitrate ions at 1423, 1382, and 1354 cm− were detectable [26]. The bands at 3420 and 1650 cm− 1 were attributable to water used in synthesis. Furthermore, sharp peaks at 1047 and 823 cm− could be ascribed to the bending modes of the nitrate ions of Ca(NO ) 4H O. Indeed, only when the thermal 3 2· 2 degradation of nitrates occurs (at a temperature beyond 350 ◦C) do calcium ions enter into the network by diffusion. Before such temperature, calcium nitrate salt coats the material network; therefore, the FTIR nitrate signals appear strong. 1 The presence of the asymmetric and symmetric stretching of P-O bonds at 1090 and 960 cm− , however, proves that a HA network was formed [27,28]. After heat treatment at 600 ◦C, another peak 1 relating to the vibrational mode of hydroxyapatite P–O groups (1033 cm− ) appeared (Figure3b), 1 whereas HA hydroxyl ions occurred at wavenumbers higher than 3420 cm− . The presence of peaks 2 1 typical of CO3 − vibrations at 1450 and 873 cm− resulted from carbonated hydroxyapatite. The HA spectrum of coating treated at 1000 ◦C is reported in Figure3c. The intensities of peaks 1 2 at 1450 and 873 cm− were evident, reflecting CO3 − released as volatile gas at higher calcination 1 1 temperatures, while the band at 3570 cm− disappeared. Finally, a red-shift from 1033 to 1040 cm− was attributable to P–O vibrational mode, because, according to XRD, peaks become narrower at this calcination temperature.

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Figure 3. ATR-FTIR spectra of HA layers after (a) untreated, (b) treated at 600 C, and (c) treated at Figure 3. ATR-FTIR spectra of HA layers after (a) untreated, (b) treated at 600 °C,◦ and (c) treated at 1000 C. 1000 °C◦ . 3.2. Bioactivity Test In spectra of untreated samples, strong signals due to the asymmetric and symmetric stretching of nitrateSEM ions images at 1423, of HA 1382 coatings, and 1354 heat cm treated−1 were detectable at 600 ◦C and[26]. 1000The bands◦C are at shown 3420 and in Figure1650 cm4.−1 Bothwere µ attributablesamples appeared to water to have used a in porous synthesis. structure Furthermore, with micropores sharp peaks of 3–4 at m 1047 in diameter and 823 oncm− the1 could surface. be During sample calcination, a partial HA carbonation occurs due to atmospheric CO as a consequence ascribed to the bending modes of the nitrate ions of Ca(NO3)2∙4H2O. Indeed, only 2when the thermal degradationof high calcium of nitratescontent in occurs the prepared (at a temperature samples. This beyond phenomenon 350 °C) do is responsiblecalcium ions for enter the Ca into/P ratio the networkof 1.50. by diffusion. Before such temperature, calcium nitrate salt coats the material network; therefore,Sol–gel the hydroxyapatite FTIR nitrate signals coating appear after strong. soaking in SBF for 7, 14, and 21 days (Figure5) showed the globularThe formationspresence of typical the asymmetric of physiological and symmetric hydroxyapatite. stretching Indeed, of P this-O bonds was more at 1090 evident and at960 21 cm days−1, however,than at 7 andproves 14 days.that a TheHA Canetwork/P ratio was was formed 1.30 for [2 the7,28 additional]. After heat presence treatment of carbonatesat 600 °C, another in simulated peak relatingbody ﬂuid to solution.the vibrational mode of hydroxyapatite P–O groups (1033 cm−1) appeared (Figure 3b), whereas HA hydroxyl ions occurred at wavenumbers higher than 3420 cm−1. The presence of peaks 3.3. Cytotoxicity Assessment typical of CO32− vibrations at 1450 and 873 cm−1 resulted from carbonated hydroxyapatite. TheCell HA viability spectrum assay of highlighted coating treated that at HA 1000 coatings °C is reported were biocompatible, in Figure 3c. The with intensities rates higher of peaks than atuncoated 1450 and substrates 873 cm− (Figure1 were 6evident,A). The reflecting latter were CO considered32− released to beas 100%volatile viable. gas at Thermal higher treatments calcination temperaturesdiﬀered slightly, while from the each band other; at 3570 it cm clearly−1 disappeared appeared. thatFinally, HA a coatingred-shift favored from 1033 attachment to 1040 cm and−1 wasproliferation, attributable although to P–O cellsvibrational were likely mode, found because in clusters, according (Figure to XRD,6B). peaks become narrower at this calcination temperature.

3.2. Bioactivity Test SEM images of HA coatings heat treated at 600 °C and 1000 °C are shown in Figure 4. Both samples appeared to have a porous structure with micropores of 3–4 µm in diameter on the surface. During sample calcination, a partial HA carbonation occurs due to atmospheric CO2 as a consequence

Coatings 2019, 9 FOR PEER REVIEW 7 of high calcium content in the prepared samples. This phenomenon is responsible for the Ca/P ratio of 1.50.

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ofCoatings high2020 calcium, 10, 203 content in the prepared samples. This phenomenon is responsible for the Ca/P ratio6 of 8 of 1.50.

Figure 4. SEM-EDS of hydroxyapatite coating after heat treatment at 600 °C and 1000 °C.

Sol–gel hydroxyapatite coating after soaking in SBF for 7, 14, and 21 days (Figure 5) showed the globular formations typical of physiological hydroxyapatite. Indeed, this was more evident at 21 days than at 7 and 14 days. The Ca/P ratio was 1.30 for the additional presence of carbonates in simulated body fluid solution.Figure 4. SEMSEM-EDS-EDS of of hydroxyapatite hydroxyapatite coating after heat treatment at 600 ◦°CC and 1000 °C.◦C.

FigureFigure 5. SEM 5. SEM-EDS-EDS of hydroxyapatite of hydroxyapatite coating coating after aftersoaking soaking in simulated in simulated bodily bodily fluids ﬂuids (SBF (SBF)) for 7, for 14, 7, 14, and and21 days. 21 days. Coatings 2019, 9 FOR PEER REVIEW 7 3.3. Cytotoxicity Assessment Cell viability assay highlighted that HA coatings were biocompatible, with rates higher than Figure 5. SEM-EDS of hydroxyapatite coating after soaking in simulated bodily fluids (SBF) for 7, 14, uncoated substrates (Figure 6A). The latter were considered to be 100% viable. Thermal treatments and 21 days. differed slightly from each other; it clearly appeared that HA coating favored attachment and proliferation3.3. Cytotoxicity, although Assessment cells were likely found in clusters (Figure 6B). Cell viability assay highlighted that HA coatings were biocompatible, with rates higher than uncoated substrates (Figure 6A). The latter were considered to be 100% viable. Thermal treatments differed slightly from each other; it clearly appeared that HA coating favored attachment and proliferation, although cells were likely found in clusters (Figure 6B).

Figure 6. (A) Results of WST-8 assay expressed as % of viability of cells seeded on hydroxyapatite heat Figure 6. (A) Results of WST-8 assay expressed as % of viability of cells seeded on hydroxyapatite treated at 600 C and 1000 C. (B) Cell morphology images of NIH-3T3 cells seeded on hydroxyapatite heat treated at◦ 600 °C and◦ 1000 °C. (B) Cell morphology images of NIH-3T3 cells seeded on heat treated at 600 C and 1000 C were acquired by Inverted Phase Contrast Brightﬁeld Zeiss Primo hydroxyapatite heat◦ treated at 600◦ °C and 1000 °C were acquired by Inverted Phase Contrast VertBrightfield Microscope Zeiss andPrimo representatively Vert Microscope shown. and representatively Un-Co = uncoated; shown. HA-CoUn-Co == uncoated;hydroxyapatite HA-Co = coated. hydroxyapatite coated. 4. Conclusions 4. ConclusionsHA was synthesized using the sol–gel method and then used for coating titanium disks. The substratesHA was were synthesized coated with using the the dip sol coating–gel method procedure, and then and used an improvementfor coating titanium in biocompatibility disks. The was demonstrated.substrates wereFT-IR coated analysis with the ofdip untreated, coating procedure 600 ◦C-,, and and an 1000 improvement◦C-treated in samplesbiocompatibility indicated was that heat demonstrated. FT-IR analysis of untreated, 600 °C-, and 1000 °C-treated samples indicated that heat treatment degraded the nitrates used in synthesis, which may adversely affect the biological response to the material. This treatment also showed a carbonation of HA at high temperatures. SEM analysis showed the film morphology of various heat treatments. Microporous structure could promote cell adhesion and proliferation. Furthermore, coated samples appeared more bioactive and biocompatible than uncoated ones.

Author Contributions: validation, M.C.; formal analysis, F.B, S.P. (Simona Piccolella), and S.P. (Severina Pacifico); data curation, M.C. and I.B.; writing—original draft preparation, M.C.; supervision, M.C. All the authors have read and approved the final manuscript.

Funding: The work was financially supported by V:ALERE 2019 grant support from Università degli studi della Campania “L. Vanvitelli” of CHIMERA.

Conflicts of Interest: The authors declare no conflicts of interest.

References

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7. Catauro, M.; Bollino, F.; Renella, R.; Papale, F. Sol–gel synthesis of SiO2–CaO–P2O5 glasses: Influence of the heat treatment on their bioactivity and biocompatibility. Ceram. Int. 2015, 41, 12578–12588. 8. Cai, S.; Wang, Y.; Lv, H.; Peng, Z.; Yao, K. Synthesis of carbonated hydroxyapatite nanofibers by mechanochemical methods. Ceram. Int. 2005, 31, 135–138.

Coatings 2020, 10, 203 7 of 8 treatment degraded the nitrates used in synthesis, which may adversely aﬀect the biological response to the material. This treatment also showed a carbonation of HA at high temperatures. SEM analysis showed the ﬁlm morphology of various heat treatments. Microporous structure could promote cell adhesion and proliferation. Furthermore, coated samples appeared more bioactive and biocompatible than uncoated ones.

Author Contributions: Validation, M.C.; formal analysis, F.B., S.P. (Simona Piccolella), and S.P. (Severina Pacifico); data curation, M.C. and I.B.; writing—original draft preparation, M.C.; supervision, M.C. All authors have read and agreed to the published version of the manuscript. Funding: The work was financially supported by V:ALERE 2019 grant support from Università degli studi della Campania “L. Vanvitelli” of CHIMERA. Conflicts of Interest: The authors declare no conflicts of interest.

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