Hydrothermal Transformation of Eggshell Calcium Carbonate into Apatite Micro-Nanoparticles: Cytocompatibility and Osteoinductive Properties

Citation: Torres-Mansilla, A.; Álvarez-Lloret, P.; Fernández-Penas, R.; D’Urso, A.; Baldión, P.A.; Oltolina, F.; Follenzi, A.; Gómez-Morales, J. Hydrothermal Transformation of Eggshell Calcium Carbonate into Apatite Micro-Nanoparticles: Cytocompatibility and Osteoinductive Properties. Nanomaterials 2023,13, 2299. https://doi.org/10.3390/ nano13162299 Academic Editor: Alexey Pestryakov Received: 17 July 2023 Revised: 5 August 2023 Accepted: 7 August 2023 Published: 10 August 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). nanomaterials Article Hydrothermal Transformation of Eggshell Calcium Carbonate into Apatite Micro-Nanoparticles: Cytocompatibility and Osteoinductive Properties Adriana Torres-Mansilla 1,2, Pedro Álvarez-Lloret 1,* , Raquel Fernández-Penas 2, Annarita D’Urso 3, Paula Alejandra Baldión4, Francesca Oltolina 3, Antonia Follenzi 3and Jaime Gómez-Morales 2,* 1Departament of Geology, University of Oviedo, 33005 Oviedo, Spain; [email protected] 2Laboratory of Crystallographic Studies, IACT-CSIC-University of Granada, Avda. Las Palmeras, n◦4, 18100 Armilla, Spain; [email protected] 3Dipartimento di Scienze della Salute, Universitàdel Piemonte Orientale, “A. Avogadro” Via Solaroli, 17, 28100 Novara, Italy; [email protected] (A.D.); [email protected] (F.O.); [email protected] (A.F.) 4Departament of Oral Health, Faculty of Dentistry, Universidad Nacional de Colombia, Bogotá111321, Colombia; [email protected] *Correspondence: [email protected] (P.Á.-L.); [email protected] (J.G.-M.) Abstract: The eggshell is a biomineral consisting of CaCO 3 in the form of calcite phase and a pervading organic matrix (1–3.5 wt.%). Transforming eggshell calcite particles into calcium phosphate (apatite) micro-nanoparticles opens the door to repurposing the eggshell waste as materials with potential biomedical applications, fulfilling the principles of the circular economy. Previous methods to obtain these particles consisted mainly of two steps, the first one involving the calcination of the eggshell. In this research, direct transformation by a one-pot hydrothermal method ranging from 100–200 ◦ C was studied, using suspensions with a stoichiometric P/CaCO 3 ratio, K 2 HPO 4 as P reagent, and eggshells particles (Ø < 50 µ m) both untreated and treated with NaClO to remove surface organic matter. In the untreated group, the complete conversion was achieved at 160 ◦ C, and most particles displayed a hexagonal plate morphology, eventually with a central hole. In the treated group, this replacement occurred at 180 ◦ C, yielding granular (spherulitic) apatite nanoparticles. The eggshell particles and apatite micro-nanoparticles were cytocompatible when incubated with MG-63 human osteosarcoma cells and m17.ASC murine mesenchymal stem cells and promoted the osteogenic differentiation of m17.ASC cells. The study results are useful for designing and fabricating biocompatible microstructured materials with osteoinductive properties for applications in bone tissue engineering and dentistry. Keywords: calcite; apatite; organic matrix; hydrothermal conversion; cytocompatibility; osteogenic differentiation 1. Introduction The eggshell (ES) of hens is a complex biomineral structure that functions as a protective barrier for the egg content, allowing the development of the chick embryo out of the uterus [ 1 ]. It is composed of a calcified shell attached to the outer surface of a polymeric membrane, known as the eggshell membrane (ESM) [2]. The shell is composed of ~96 wt.% calcium carbonate (CaCO 3 ) in the form of calcite, an intracrystalline occluded organic matrix (OM, 1–3.5 wt.%), and the remaining components include Mg, P, H 2 O, and other trace elements [ 2 ]. The OM is composed of proteins, glycoproteins, and proteoglycans. Among these organic components, it is worth mentioning egg white proteins such as ovalbumin, lysozyme and ovotransferrin, ubiquitous proteins (e.g., osteopontin and clusterin), and organic constituents unique to the process of shell calcification. This latter group comprises dermatan and keratan proteoglycans, ovocleidins Nanomaterials 2023,13, 2299. https://doi.org/10.3390/nano13162299 https://www.mdpi.com/journal/nanomaterials Nanomaterials 2023,13, 2299 2 of 18 and ovocalyxins [ 1 ]. A mass spectrometry-based high-throughput proteomic study of the acid-soluble OM of the chicken eggshell identified more than 500 proteins in the shell [ 3 ]. This OM content is fundamental in eggshell calcification and formation, leading to a highly structured material with intrinsic porosity. The organic components have been proven to control the nucleation and growth of calcium carbonate during in vitro experiments, yielding crystalline morphologies similar to those of the shell formed in vivo [4,5]. ES is an abundant source of naturally occurring carbonate formed from biologically controlled biomineralization processes. However, these materials and their associated ESM represent waste by-products discarded in the food industry. This waste is commonly considered useless and is often disposed of in landfills without any pretreatment. This waste management is not a desirable practice in view of the environmental odor from biodegradation. The disposal is ecologically harmful and implies the loss of potentially valuable biomaterials [ 6 , 7 ]. According to estimates [ 8 ], the amount of ES discarded in 2018 was close to 8.5 million tons and represented the fifteenth largest source of pollution in the food industry. Reusing this by-product would reduce the economic and environmental burden related to its processing. Different proposals to transform ES waste into valuable materials include its use as a low-cost adsorbent for the removal of ionic pollutants from the aqueous solution [ 9 , 10 ], as a solid base catalyst used for biodiesel pollutant minimization [ 11 ], as a dietary calcium supplement in human and animal food, or plants fertilizer [ 12 ], or as an environmentally friendly raw material to produce calcium phosphate apatite (Ap) bioceramics [13]. Apatites are the main inorganic component of bones and teeth in mammals. Synthetic nanocrystalline apatites (Ap NPs) exhibit high biocompatibility, bioactivity, and a relatively high bioresorbability in biological media. Their synthesis and characterization methods can be approached from many different perspectives and offer numerous technological and industrial applications [ 14 , 15 ]. The modulation of their physicochemical properties has allowed the use of Ap NPs in advanced biomedical applications, such as bone tissue regeneration [ 16 ], as nanocarriers for drug delivery [ 17 ], or as luminescent probes in bioimaging [ 18 – 20 ], among others. In the field of bone tissue regeneration, the use of other inorganic nanoparticles, for example, platinum nanoparticles (Pt NPs), has been reported to enhance the osteogenic differentiation of human dental follicle stem cells [ 21 ]. However, particles based on Ag NPs present advantages in terms of compositional compatibility with calcified tissues. ES-derived Ap particles have been obtained mainly by two-step processes involving the calcination of powdered ES to CaO and CO 2 at temperatures above 800–900 ◦ C, followed by a reaction of CaO with phosphoric acid [ 22 – 24 ] or with tricalcium phosphate and (NH 4 ) 2 HPO 4 in hydrothermal conditions at high temperatures [ 25 , 26 ]. These multi-step processes involving a calcination step fully degrade or destroy the intracrystalline OM and any surface organic residue of the ES powder. Additionally, these processes are energetically expensive and environmentally poorly sustainable. Recently, a “one-pot” method to directly transform biogenic CaCO 3 particles into apatite micro-nanoparticles under mild hydrothermal conditions was set up [ 27 ]. The method avoids the calcination step as well as the use of acids and other additives. In processes of mineral replacement from biogenic calcium carbonate under mild conditions, the organic matter content can play a critical role. Bleaching with sodium hypochlorite (NaClO) is a common practice to remove the surface organic/tissue residues of biominerals [ 28 ]. The treatment must not affect the intracrystalline occluded OM. In this research, we investigated the transformation of bleached (NaClO-treated) powdered ES into Ap micro/nanoparticles with cytocompatible and osteoinductive properties for osteogenic applications using the one-pot hydrothermal method. Additionally, we investigated the transformation of non-bleached powdered ES (NaClO-untreated) into Ap to gain insight into the role of the surface organic residues in the transformation process as well as on the physicochemical and morphological characteristics of the obtained Ap particles. The aim is to contribute to developing a strategy for reusing this biogenic waste Nanomaterials 2023,13, 2299 3 of 18 in particles of biomedical interest by setting up a cost-effective (with a reduced number of processing operations) and eco-friendly procedure by means of a low-temperature hydrothermal method. 2. Experimental Section 2.1. Materials and Methods The eggshells were collected from hen eggs. The ESM was carefully removed and discarded. The remaining CaCO 3 shells were crushed and divided into two groups. The first group was treated with NaClO 5% v/vfor 24 h, washed with tap water, dried in air, and labeled ES(t); the second one was not bleached; it was only washed with tap water and air-dried. This untreated group was labeled ES(u). Both ES groups were ground in an agate mortar and sieved to particle sizes Ø < 50 µm. The experiments were performed inside a Memmert UNB 200 oven (Memmert, Schwabach, Germany) with circulating forced air, using a specially designed aluminum box that contained four 10 mL PTFE tubes. The box had an aluminum cap coated with PTFE to close the ensemble. The tubes were filled to 70% with suspensions of either ES(t) or ES(u) and K 2 HPO 4 in stoichiometric K 2 HPO 4 /CaCO 3 ratios with respect to hydroxyapatite (0.6). All suspensions were prepared with ultrapure Milli-Q water (resistivity 18.2 M. Ω .cm, Millipore, Merck, Burlington, MA, USA) and analytical grade reactants ( ≥ 97–99%; Sigma Aldrich, Darmstadt, Germany). Hydrothermal reactions were carried out between 100 and 200 ◦ C ( ∆ 20 ◦ C) for 7 days. Then, the precipitates were washed by centrifugation with deionized water (3 cycles, 9000 rpm each) to remove unreacted species and freeze-dried under vacuum (3 millibars) at −50 ◦C overnight. 2.2. Physicochemical Characterization The physicochemical characterization was performed by X-ray diffraction (XRD), highresolution scanning electron microscopy (HRSEM), energy dispersive X-ray spectroscopy microanalysis (EDX), Fourier transform infrared-attenuated total reflectance (FTIR-ATR) and Raman spectroscopies, thermogravimetry (TGA), dynamic light scattering (DLS) and ζ-potential against pH. XRD data were obtained using a PHILIPS X’PERT PRO X-ray diffractometer (Almelo, The Netherlands). For diffraction experiments, the working conditions were Cu K α1 radiation ( λ = 1.54059 Å) at 45 kV and 40 mA. XRD patterns were collected from 4 ◦ to 60 ◦ (2 θ ), with a 2 θ scan step of 0.007 ◦ and a counting time of 1 s per step. Phase identification was performed by matching the experimental diffraction patterns with those included in the crystal structure information provided by the American Mineralogist Crystal Structure Database (AMCSD). The average crystallite sizes were determined as Lvol-IB values (i.e., volume-weighted mean crystallite sizes calculated by FWHM and integral amplitude) using TOPAS V.4.2 software. High-resolution scanning electron microscopy was performed with an AURIGA FIBFESEM microscope (Carl Zeiss SMT Inc., Danvers, MA, USA) with an accelerating voltage of 5 kV, coupled to an X-ray energy-dispersive spectrometer from Oxford Instruments for chemical micro-analysis. The samples were carbon-coated prior to observation. FTIR-ATR characterization was performed with an Invenio R FTIR spectrometer (Bruker, Billerica, MA, USA) equipped with an attenuated total reflectance (ATR) accessory of a diamond crystal. Spectra were recorded within the wavenumber range from 4000 cm −1 to 400 cm −1 at a resolution of 2 cm −1 . Raman spectra were recorded with a LabRAMHR spectrometer (Jobin–Yvon, Horiba, Kyoto, Japan). The excitation line was provided by a diode laser emitting at a wavelength of 532 nm. A Peltier-cooled charge-coupled device (CCD) (1064 ×256 pixels) was used as the detector. Crystal size distributions (CSD, mass) and ζ -potential were measured with a Malvern Zetasizer Nano ZS analyzer (Malvern Instruments Ltd., Malvern, UK) using, respectively, quartz and disposable polystyrene cuvettes filled with aqueous suspensions of ~0.5 mg/mL. Nanomaterials 2023,13, 2299 4 of 18 For ζ -potential versus pH measurements, we used 0.1 M HCl and 0.1 M NaOH as titrant solutions without any additional electrolytes. Thermogravimetric analyses (TGA-DSC3+, Mettler Toledo, Columbus, OH, USA) were performed under air between 30 and 950 ◦ C at a constant 20 ◦ C /min heating rate. The mass loss during heating was used to determine the water (up to 180 ◦ C), organic matter (range 180 to 550 ◦ C), and mineral (above 550 ◦ C) contents, within which the fraction of carbonate component (between 55 to 850 ◦C) was also calculated. 2.3. Biological Tests 2.3.1. Cell Cultures The human osteosarcoma cell line MG-63 (ATCC ® CRL–1427 ™ ) was maintained in Dulbecco modified Eagle’s medium (DMEM) (Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal bovine serum (FBS), antibiotic solution (streptomycin 100 µ g/mL and penicillin 100 U/mL, Sigma-Aldrich, Milan, Italy) and 2 mM L-glutamine (complete medium). M17.ASC cells (a spontaneously immortalized mouse mesenchymal stem cell clone from subcutaneous adipose tissue) [ 29 ] were cultured in Claycomb medium (Sigma-Aldrich, Milan, Italy), supplemented as described above. Cells were incubated at 37 ◦ C in a humidified atmosphere with 5% CO 2 , and they were regularly split when sub-confluent at a ratio of 1:8 and 1:10 for MG-63 and m17.1ASC, respectively. 2.3.2. Cytocompatibility MG-63 cells (5 × 10 3 /well) and m17.ASC (2 × 10 3 /well) were seeded in 96-well plates, and 24 h after, different concentrations (ranging from 0.1 to 100 µ g/mL) of particles were added in 100 µ L of fresh complete medium. The particles used in this study were ES(u), ES(t), Ap(t)-160 ◦ C and Ap(t)-200 ◦ C. After 72 h of incubation, cell viability was evaluated by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT, Sigma) colorimetric assay as described in Cano Pláet al. [ 19 ]. Briefly, 20 µ L of MTT solution (5 mg/mL) in phosphate buffer saline (PBS) was added to each well, and the plate was incubated at 37 ◦ C for 2 h. Afterward, supernatants were carefully aspirated, and then 125 µ L of 0.2 N HCl in isopropanol was added to dissolve formazan crystals. Then, 100 µ L were removed, and the optical density was measured in a multi-well reader (2030 Multilabel Reader Victor TM X4, Perkin Elmer, Waltham, MA, USA) at 570 nm. Hydrogen peroxide (1 µ M) was used as control of toxicity. The absorbance value of untreated cells was taken as 100% viability, and values obtained from cells undergoing the different treatments were compared to this value. Experiments were performed at least three times using 3 replicates for each sample. 2.3.3. Osteogenic Differentiation of m17.ASC m17.ASC cells were seeded onto 12-well plates at a density of 1 × 10 4 cells per well. After 24 h, they were treated for 14 days with a concentration of 25 µ g/mL of Es(u) and apatite NPs transformed at 200 ◦ C to evaluate the osteoinductive potential effects of NPs. Cells were compared to a positive control group that was cultured for the same time with an osteogenic medium (Ob) containing DMEM, FBS 10%, 50 µ g/mL ascorbic acid (MW 176.12), 10 mM β -glycerophosphate (MW 216.04), and 10 nM dexamethasone (Sigma-Aldrich). The culture medium was replaced every 3 days during the treatment period, as previously described in [29]. 2.3.4. Alkaline Phosphatase (ALP) Staining and Quantitative Analysis The osteogenic differentiation was estimated by alkaline phosphatase (ALP) staining and quantified with ImageJ 1.48v software as described in Dupont et al. [ 30 ]. Briefly, after 14 days of culture with the particles, cells were washed three times with PBS, fixed with 4% paraformaldehyde (4% PFA; Sigma-Aldrich) for 15 min, and stained with an alkaline phosphatase detection kit (Millipore, Merck Millipore, Milan, Italy) according to the manufacturer’s protocol. Untreated cells and cells differentiated with the osteogenic Nanomaterials 2023,13, 2299 5 of 18 medium were used as a negative and positive control, respectively. Images were acquired under optical microscopy at 200 × magnification. For ALP quantification, the intensity of violet staining was estimated as the integrated density (INT.DEN.) by ImageJ analysis. This value was normalized to the number of cells for each picture and was expressed as an arbitrary unit (A.U.). Experiments were performed at least three times. 2.3.5. Alizarin Red Staining (ARS) and Quantitative Analysis To determine the calcium deposition in m17.ASC cells, as a consequence of the osteogenic differentiation after a 14-day treatment with osteogenic medium or particles, Alizarin Red S staining was carried out as described in Sutthavas et al. [ 31 ]. Briefly, cells were washed with PBS pH 7.2, fixed with 2% PFA (2 wt% in PBS), and then stained with Alizarin Red S solution (40 mM), pH 4.1, for 30 min at room temperature. Afterward, the cells were washed thrice with bi-distilled water to remove the non-specific precipitation, and then samples were analyzed and photographed by optical microscopy at 200 × . To quantify the staining, mineralized deposits were dissolved in 10% acetic acid (SigmaAldrich) for 30 min, and then 150 µ L of each sample was collected in a 96-well plate to measure their optical density in a multi-well reader (2030 Multilabel Reader Victor TM X4, Perkin Elmer) at 405 nm. Experiments were performed at least three times using 3 replicates for each sample. 2.3.6. Statistical Analysis Data were statistically analyzed and are expressed as mean ± standard deviation of at least three replicates. Statistical analyses were performed using one-way ANOVA with Bonferroni’s post-test for grouped analyses using GraphPad Prism version 7.0 for Mac, GraphPad Software (GraphPad Prism, San Diego, CA, USA). Statistical differences between the treatments were considered significant when p values were p< 0.05 (*), p< 0.01 (**), p< 0.001 (***), and p< 0.0001 (****). 3. Results 3.1. Crystallographic, Morphological, Compositional, and Spectroscopic Features In both ES groups, the starting material (Figure 1, bottom) was identified as calcite, depicted by its characteristic XRD pattern (PDF 00-005-0586). The diffractograms showed the most intense reflection at 2 θ = 29.3 ◦ (plane 104). The samples displayed a nanogranular texture characteristic of biological calcite [ 32 ]. Treatment with NaClO and milling neither induced the formation of additional crystalline phases nor seemed to modify the particle morphology. After hydrothermal reaction (Figure 1a) at 100 ◦ C, the ES(t) group showed the partial transformation from ES(t) into Ap(t), characterized by the emerged reflections at 2 θ = 25.87 ◦ (002) and those at 31.77 ◦ (211), 32.19 ◦ (112), 32.90 ◦ (300), and 33.97 ◦ (202), respectively (PDF 01-1008). The total transformation from ES(t) into Ap(t) occurred at 180 and 200 ◦ C, witnessed by the lack of the (104) calcite reflection in these diffractograms. The average crystallite size determined by XRD (i.e., the crystalline domain size) of both ES(t) and Ap(t)- 200 ◦ C were 190 and 80 nm, respectively. SEM pictures of Ap samples showed crystals with globular morphologies forming larger aggregates (Figure 1(ai,aii)), similar to those found by other authors [ 33 ]. The EDX microanalysis of the Ap(t)-200 ◦ C sample yielded 39.30 wt.% Ca, 17.85 wt.% P, 39.8 wt.% O, 2.65 wt.% K, and 0.65 wt.% Mg, with (Ca+Mg)/P = 1.74. Note that in this analysis, the C was ruled out since we used C to metalize the samples prior to SEM observation, but it did not influence the calculation of the (Ca+Mg)/P molar ratio. At intermediate temperatures (i.e., 100–160 ◦ C), the SEM images showed globular morphologies due to both phases ES(t) and Ap(t) being indistinguishable (Figure 1(aiii,aiv)). Nanomaterials 2023,13, 2299 6 of 18 Nanomaterials 2023, 13, x FOR PEER REVIEW 6 of 18 Figure 1. XRD patterns and SEM images of (a) ES(t) and (b) ES(u) before and after being submitted to hydrothermal conversion between 100 and 200 °C. In parallel, in the ES(u) group, the sample submitted to hydrothermal conversion at 100 °C did not undergo transformation (Figure 1b), but at 120 and 140 °C, the transformation was very high, and at 160–200 °C, the ES(u) sample transformed completely. The EDX composition of the Ap(u)-200 °C sample consisted of 38.9 wt.% Ca, 18.0 wt.% P, 39.9 wt.% O, 1.5 wt.% K, and 0.5 wt.% Mg, yielding a (Ca+Mg)/P mol ratio of 1.67. The average crystallite sizes of both ES(u) and Ap(u)-200 °C were 19 nm and 55 nm, respectively. The larger crystallite size of ES(t) compared to ES(u) revealed the growth of the CaCO 3 crystalline domain size when removing the surface OM of the sample. Besides this observation, the notable finding was that many apatite single crystals displayed a hexagonal plate morphology, whose width and height were up to 1.5 µm and 0.75 µm (Figure 1(bii)), respectively, and sometimes showed a centric hole (Figure 1(biii)). These morphologies strongly differed from that obtained in the ES(t) series. Figure 2b–d show the high-resolution images of these remarkable morphologies obtained at 120, 160 and 200 °C, compared with the spherulitic one obtained in the ES(t) series (Figure 2a). The finding highlights the strong impact of the surface OM of the ES(u) on the mineral replacement reaction and on the morphological aspect of the Ap particles. At 200 °C, however, the morphology became more granular, as in the homologous experiment with the ES(t) group. The average particle sizes for the Ap(u)-200 °C and Ap(t)-200 °C samples measured from SEM images were 87.5 ± 13.8 and 131.3 ± 10.6 nm, respectively. Figure 1. XRD patterns and SEM images of ( a ) ES(t) and ( b ) ES(u) before and after being submitted to hydrothermal conversion between 100 and 200 ◦C. In parallel, in the ES(u) group, the sample submitted to hydrothermal conversion at 100 ◦ C did not undergo transformation (Figure 1b), but at 120 and 140 ◦ C, the transformation was very high, and at 160–200 ◦ C, the ES(u) sample transformed completely. The EDX composition of the Ap(u)-200 ◦ C sample consisted of 38.9 wt.% Ca, 18.0 wt.% P, 39.9 wt.% O, 1.5 wt.% K, and 0.5 wt.% Mg, yielding a (Ca+Mg)/P mol ratio of 1.67. The average crystallite sizes of both ES(u) and Ap(u)-200 ◦ C were 19 nm and 55 nm, respectively. The larger crystallite size of ES(t) compared to ES(u) revealed the growth of the CaCO 3 crystalline domain size when removing the surface OM of the sample. Besides this observation, the notable finding was that many apatite single crystals displayed a hexagonal plate morphology, whose width and height were up to 1.5 µ m and 0.75 µ m (Figure 1(bii)), respectively, and sometimes showed a centric hole (Figure 1(biii)). These morphologies strongly differed from that obtained in the ES(t) series. Figure 2b–d show the high-resolution images of these remarkable morphologies obtained at 120, 160 and 200 ◦ C, compared with the spherulitic one obtained in the ES(t) series (Figure 2a). The finding highlights the strong impact of the surface OM of the ES(u) on the mineral replacement reaction and on the morphological aspect of the Ap particles. At 200 ◦ C, however, the morphology became more granular, as in the homologous experiment with the ES(t) group. The average particle sizes for the Ap(u)-200 ◦ C and Ap(t)-200 ◦ C samples measured from SEM images were 87.5 ±13.8 and 131.3 ±10.6 nm, respectively. Nanomaterials 2023,13, 2299 7 of 18 Nanomaterials 2023, 13, x FOR PEER REVIEW 7 of 18 Figure 2. SEM images of (a) Ap(t)-180 °C, (b) Ap(u)-120 °C, (c) Ap(u)-160 °C, and (d) Ap(u)-200 °C. The thermogravimetric characterization of ES(u), ES(t), Ap(u)-200 °C and Ap(t)-200 °C (Figure 3a,b) showed that the NaClO treatment removed approximately 50% of the OM content of ES(t) with respect to the ES(u) sample, most likely all surface organic components. Both samples had different mineral content, approximately 98% and 95%. On the other hand, the transformed samples, Ap(u)-200 °C and Ap(t)-200 °C, showed a slight reduction in OM content and a significant removal of the mineral carbonate (~2–5%) component (see Table 1). Table 1. Chemical composition (wt.%) obtained from TG data analyses. Temperature ranges considered for the calculation of each component: water (up to 180 °C), organic matter (180–550 °C), carbonate mineral (550–850 °C), and total mineral (>550 °C) contents. ES(u) ES(t) Ap(u)-200 °C Ap(t)-200 °C Water 1.64 0.30 0.42 0.52 OM 3.09 1.55 1.07 1.26 Mineral 95.27 98.15 98.51 98.22 Carbonate (550–850 °C) - - 1.92 5.65 Figure 2. SEM images of ( a ) Ap(t)-180 ◦ C, ( b ) Ap(u)-120 ◦ C, ( c ) Ap(u)-160 ◦ C, and ( d ) Ap(u)-200 ◦ C. The thermogravimetric characterization of ES(u), ES(t), Ap(u)-200 ◦ C and Ap(t)-200 ◦ C (Figure 3a,b) showed that the NaClO treatment removed approximately 50% of the OM content of ES(t) with respect to the ES(u) sample, most likely all surface organic components. Both samples had different mineral content, approximately 98% and 95%. On the other hand, the transformed samples, Ap(u)-200 ◦ C and Ap(t)-200 ◦ C, showed a slight reduction in OM content and a significant removal of the mineral carbonate (~2–5%) component (see Table 1). Table 1. Chemical composition (wt.%) obtained from TG data analyses. Temperature ranges considered for the calculation of each component: water (up to 180 ◦ C), organic matter (180–550 ◦ C), carbonate mineral (550–850 ◦C), and total mineral (>550 ◦C) contents. ES(u) ES(t) Ap(u)-200 ◦C Ap(t)-200 ◦C Water 1.64 0.30 0.42 0.52 OM 3.09 1.55 1.07 1.26 Mineral 95.27 98.15 98.51 98.22 Carbonate (550–850 ◦C) - - 1.92 5.65 The ATR-FTIR spectra also evidenced the mineral conversion (Figure 4). Both ES(t) and ES(u) particles (Figure 4a,c, bottom) presented the calcite CO 3 vibrations ν4 (asymmetric stretching) at 712 cm −1 , at ~873 cm −1 the ν2 out-of-plane bending, and at ~1410 cm −1 the ν3 in-plane bending [ 34 – 36 ]. The ~1089 cm −1 peak (symmetric stretching, ν1 ) was not pronounced or identified in the figure. The peak position ν3 shifted to higher wavenumbers with respect to that of geogenic calcite (see Supporting Information, Figure S1) because of the presence of Mg [ 37 ]. The 1088 cm −1 ( ν1 CO 3 ) was, however, active in Raman, being the most intense to identify the calcite of ES (Figure 4b,d, bottom). Besides these peaks, the Nanomaterials 2023,13, 2299 8 of 18 FTIR spectrum of ES(u) also showed a tiny band at 1650 cm −1 , even smaller in ES(t), which corresponded to the amide II of side chains of proteins. Nanomaterials 2023, 13, x FOR PEER REVIEW 8 of 18 Figure 3. Thermogravimetric analysis (TGA) showing the heating curves for (a) ES(u), ES(t), Ap(u)-200 °C, and Ap(t)-200 °C up to 850 °C. (b) Idem up to 550 °C. The ranges of temperatures for the calculation of each component were the following: water (up to 180 °C), organic matter (180–550 °C), carbonate mineral (550–850 °C) and total mineral content (>550 °C) content. The ATR-FTIR spectra also evidenced the mineral conversion (Figure 4). Both ES(t) and ES(u) particles (Figure 4a,c, bottom) presented the calcite CO 3 vibrations ν 4 (asymmetric stretching) at 712 cm −1 , at ~873 cm −1 the ν 2 out-of-plane bending, and at ~1410 cm −1 the ν 3 inplane bending [34–36]. The ~1089 cm −1 peak (symmetric stretching, ν 1 ) was not pronounced or identified in the figure. The peak position ν 3 shifted to higher wavenumbers with respect to that of geogenic calcite (see Supporting Information, Figure S1) because of the presence of Mg [37]. The 1088 cm −1 (ν 1 CO 3 ) was, however, active in Raman, being the most intense to identify the calcite of ES (Figure 4b,d, bottom). Besides these peaks, the FTIR spectrum of ES(u) also showed a tiny band at 1650 cm −1 , even smaller in ES(t), which corresponded to the amide II of side chains of proteins. When submitting samples to hydrothermal conversion, in both groups (Figure4a,c) appeared the characteristic vibrational modes of PO 4 groups of the apatite, i.e., at ~1020 cm −1 , the asymmetric stretching mode (ν 3 ); at ~960 cm −1 the symmetric stretching (ν 1 ), at ~600 and ~560 cm −1 , the bending modes (ν 4 ), and at ~470 cm −1 , the ν 2 PO 4 [38]. In addition, the vibrational modes ν 2 CO 3 at around 873 cm −1 and ν 3 CO 3 at about 1410 cm −1 remained in the transformed samples, though with very low intensity, indicating that the apatites were CO 32— substituted. In this case, the ν 3 CO 3 appeared to split into two bands, at ~1410 and ~1460 cm −1 , as usually observed in CO 32— substituted apatites [20]. A deconvolution was performed on the low-intensity absorption band centered at 873 cm −1 (Figure S2), revealing three different sub-bands located at ~880, ~873, and ~870 cm −1 attributed to A-type (CO 32— replacing OH - ), B-type (CO 32— replacing PO 43- ) and labile CO 32— species located at the surface of the nanoparticles [38,39]. The degree of carbonation was also estimated using a quantification methodology [38] that compared the relative intensity of the FTIR ν 2 CO 3 carbonate bands relative to the ν 1 - ν 3 PO 4 phosphate bands. Following this methodological approach, the overall carbonation degree was close to 5 wt.% for Ap(t) and 3.5 wt.% for Ap(u) obtained at 200 °C, in the range of that calculated by TG. Finally, the Raman spectra (Figure 4b,d) also showed the presence of the most intense mode ν 1 PO 4 at 960 cm −1 and the ν 3 at 1070 cm −1 , while the ν 1 CO 3 at 1088 cm −1 progressively disappeared when increasing the temperature. Figure 3. Thermogravimetric analysis (TGA) showing the heating curves for ( a ) ES(u), ES(t), Ap(u)- 200 ◦ C, and Ap(t)-200 ◦ C up to 850 ◦ C. ( b ) Idem up to 550 ◦ C. The ranges of temperatures for the calculation of each component were the following: water (up to 180 ◦ C), organic matter (180–550 ◦ C), carbonate mineral (550–850 ◦C) and total mineral content (>550 ◦C) content. When submitting samples to hydrothermal conversion, in both groups (Figure 4a,c) appeared the characteristic vibrational modes of PO 4 groups of the apatite, i.e., at ~1020 cm −1 , the asymmetric stretching mode ( ν3 ); at ~960 cm −1 the symmetric stretching ( ν1 ), at ~600 and ~560 cm −1 , the bending modes ( ν4 ), and at ~470 cm −1 , the ν2 PO 4 [ 38 ]. In addition, the vibrational modes ν2 CO 3 at around 873 cm −1 and ν3 CO 3 at about 1410 cm −1 remained in the transformed samples, though with very low intensity, indicating that the apatites were CO 32— substituted. In this case, the ν3 CO 3 appeared to split into two bands, at ~1410 and ~1460 cm −1 , as usually observed in CO 32— substituted apatites [ 20 ]. A deconvolution was performed on the low-intensity absorption band centered at 873 cm −1 (Figure S2), revealing three different sub-bands located at ~880, ~873, and ~870 cm −1 attributed to A-type (CO 32— replacing OH - ), B-type (CO 32— replacing PO 43- ) and labile CO 32— species located at the surface of the nanoparticles [ 38 , 39 ]. The degree of carbonation was also estimated using a quantification methodology [ 38 ] that compared the relative intensity of the FTIR ν2 CO 3 carbonate bands relative to the ν1 - ν3 PO 4 phosphate bands. Following this methodological approach, the overall carbonation degree was close to 5 wt.% for Ap(t) and 3.5 wt.% for Ap(u) obtained at 200 ◦ C, in the range of that calculated by TG. Finally, the Raman spectra (Figure 4b,d) also showed the presence of the most intense mode ν1 PO 4 at 960 cm −1 and the ν3 at 1070 cm −1 , while the ν1 CO 3 at 1088 cm −1 progressively disappeared when increasing the temperature. Nanomaterials 2023,13, 2299 9 of 18 Nanomaterials 2023, 13, x FOR PEER REVIEW 9 of 18 Figure 4. ATR-FTIR and Raman spectra of (a,b) ES(t) and (c,d) ES(u) before and after being submitted to hydrothermal conversions between 100 °C and 200 °C. 3.2. Crystal Size Distribution and ζ-Potential Versus pH of ES and Derived Ap Particles The crystal size distribution and ζ-potential of particulate suspensions at pHs of physiological interest (i.e., 7.4 simulating the pH in blood [40], 7.2–7.4 in bone tissue [41], and 5.6–7 in a tumor microenvironment or inflamed tissue [42,43] were essential characteristics to evaluate the possible biomedical applications of ES and derived Ap micro-nanoparticles as nanocarriers for drug delivery, or as implantable materials in bone tissue regeneration. Both ES(u) and ES(t) were composed of aggregates of nanoparticles showing a multimodal distribution (Figure 5a). The CSD of ES(u) showed particle sizes of around 50 nm and aggregates of up to 5.5 µm, while ES(t) was composed of particles with a median size of 68 nm and aggregates of 615 nm. The plot as cumulative volume-based distribution (Figure 5b) revealed the percentiles D 10 , D 50, and D 90 of the distribution. In these samples, D 10 was 44 and 88 nm, respectively, close to the individual particle size, while D 50 (the median of the distribution) and D 90 were affected by aggregation. On the other hand, Ap(u)-200 °C and Ap(t)- 200 °C showed D 10 values of 116 and 202 nm and D 50 values of 574 and 742 nm, respectively, Figure 4. ATR-FTIR and Raman spectra of ( a , b ) ES(t) and ( c , d ) ES(u) before and after being submitted to hydrothermal conversions between 100 ◦C and 200 ◦C. 3.2. Crystal Size Distribution and ζ-Potential Versus pH of ES and Derived Ap Particles The crystal size distribution and ζ -potential of particulate suspensions at pHs of physiological interest (i.e., 7.4 simulating the pH in blood [ 40 ], 7.2–7.4 in bone tissue [ 41 ], and 5.6–7 in a tumor microenvironment or inflamed tissue [ 42 , 43 ] were essential characteristics to evaluate the possible biomedical applications of ES and derived Ap micro-nanoparticles as nanocarriers for drug delivery, or as implantable materials in bone tissue regeneration. Both ES(u) and ES(t) were composed of aggregates of nanoparticles showing a multimodal distribution (Figure 5a). The CSD of ES(u) showed particle sizes of around 50 nm and aggregates of up to 5.5 µ m, while ES(t) was composed of particles with a median size of 68 nm and aggregates of 615 nm. The plot as cumulative volume-based distribution (Figure 5b) revealed the percentiles D 10 , D 50, and D 90 of the distribution. 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