Bridging the Effects of Noncontact Temperature Sensing and Cellular Biofunctionality in Nanosized Dysprosium(III)‐Doped Fluorapatite

TL;DR

This paper explores how dysprosium-doped fluorapatite nanomaterials can sense temperature and support cell activity, making them promising for biomedical applications.

Contribution

The study introduces a multifunctional nanomaterial that combines temperature sensing and bioactivity for regenerative medicine.

Findings

Dy³⁺-doped fluorapatite shows stable temperature sensitivity through photoluminescence.

The material exhibits antimicrobial activity against six microbial species.

It supports progenitor cell activity and shows biocompatibility in in vitro assays.

Abstract

Thermal imaging plays a pivotal role in distinguishing distinct cellular states, assessing dynamic cellular activity in real‐time health monitoring, and advancing the design of biofunctional materials for tissue engineering applications. The investigated photoluminescence characteristics of Dy3⁺ ion‐doped fluorapatite demonstrate that this material offers stable temperature sensitivity. Our findings support the strategic design of next‐generation nanomaterials for regenerative medicine and tissue engineering by elucidating key cellular interactions. Furthermore, our study has begun to address the impact of Dy3⁺ ion‐doped nanomaterials on progenitor cell activity, providing valuable insights into their potential applications. A comprehensive description of photoluminescence characterization, including the LIR and SR parameters, is provided to highlight its high thermal sensing potential. The antimicrobial activity of Dy3⁺ ion‐doped fluorapatite is confirmed using the well‐diffusion method against six commonly encountered microbial species. In vitro assays on hBMSCs included, among others, the determination of apoptosis profiles and metabolic potential through cytometric assays and molecular analyses (RT‐qPCR and Western blot). These findings underscore the potential of Dy3⁺ ion‐doped fluorapatite as a biocompatible material for biomedical applications while emphasizing the importance of further understanding its mechanism of action.

Multifunctional dysprosium(III) ion–doped fluorapatite nanomaterials combine bioactivity with photoluminescent temperature sensing. The biological studies reveal interactions with progenitor cells, highlighting potential applications in regenerative medicine and tissue engineering, while temperature‐dependent emission of Dy³⁺ ions enables reliable optical temperature sensors.

Figures

X‐ray diffraction pattern of Ca10‐xDyx(PO4)6F2, where x = 0.1–0.7, heat‐treated at 600°C.

The growth inhibition zone was tested for a Dy3+‐doped apatite. RS – reference stain, CI – clinical isolate.

The effect of Dy3⁺ion‐doped nanosized apatite‐type materials on the metabolism of human bone marrow stromal cells (hBMSCs). Cellular metabolism was assessed using the Alamar Blue assay, with results expressed as the metabolic factor relative to the activity of cells in the control cultures (expressed as 1). A comparative analysis was conducted using results from four independent experiments. The data are displayed as bar graphs, representing the mean ± standard deviation (SD). Statistical significance is denoted by asterisks, where *indicates p‐value  < 0.05; p‐value < 0.01; * p‐value < 0.001, and indicates p‐value < 0.0001, while “ns” denotes non‐significant differences.

The effect of Dy3⁺ion‐doped nanosized apatite‐type materials on the morphology and growth pattern of human bone marrow stromal cells (hBMSCs). Epifluorescence microscopy was used to monitor potential changes in hBMSCs culture morphology resulting from biomaterial exposure. The nuclei were stained with DAPI (blue signal), while the actin cytoskeleton was counterstained with phalloidin Atto‐488 (green signal). The red signal corresponds to the nanomaterials doped with Dy3⁺ ions. The cells were visualized at 100‐fold magnification. The scale bar (see in control cultures) equals 200 µm.

The influence of Dy3+‐ doped nanocomposites on the viability of human bone marrow stromal cells (hBMSCs). The representative images (panel a) show the distribution of cells based on Annexin FITC‐A staining, allowing distinguishing of different cell populations: live cells (LL—lower left quadrant), early apoptotic cells (LR—lower right quadrant), late apoptotic cells (UL—upper left quadrant), and necrotic cells (UR—upper right quadrant) (a). A comparative analysis of apoptotic cell populations was conducted (b‐d), with significant differences indicated by asterisks (* p < 0.001; p < 0.01; * p < 0.05), while nonsignificant differences are denoted as ns (b).

The influence of Dy3+ ion‐doped nanosized apatite‐type materials on the expression of key apoptosis‐ and cell cycle‐regulating genes. The analysis included the determination of pro‐apoptotic markers, i.e., BAD and BAX, and anti‐apoptotic markers, i.e., BCL‐2 (c) and BCL‐XL (g). Moreover, the BAX/BCL‐2 ratio was determined to assess the balance between pro‐ and anti‐apoptotic signaling (d). Additionally, mRNA levels of p53 (e) and p21 (f) were analyzed to evaluate the activation of cell cycle regulatory pathways. A comparative analysis was conducted to evaluate the significance of the observed expression patterns. Statistically significant differences were indicated as follows: * p‐value < 0.05; p‐value < 0.01; * p‐value < 0.001, and indicates p < 0.0001, while nonsignificant differences are denoted as ns (b).

The influence of Dy3+‐ doped nanocomposites on mitochondrial membrane potential (ΔΨm ) of human bone marrow stromal cells (hBMSCs). The representative images (panel a) show the distribution of cells based on the fluorescence intensity of JC‐1 dye, indicating changes in ΔΨm . Cells with high ΔΨm exhibit red fluorescence due to J‐aggregates, whereas cells with depolarized mitochondria show green fluorescence from JC‐1 monomers.(a). A comparative analysis of cells with high and low ΔΨm was performed (b‐c), with significant differences indicated by asterisks (* p‐value  < 0.05; * p‐value < 0.001 and indicates p < 0.0001) while nonsignificant differences are denoted as ns.

The influence of Dy3+‐doped nanocomposites on the expression of regulators of mitochondrial dynamics, mitophagy, and mitochondrial quality control. The analysis included mRNA expression of MFN1 (a), PINK1 (c), PARKIN (f), FIS1 (h), and miR‐19a‐3p (i). Protein‐level assessment involved MFN1, PINK1, and PARKIN abundance and intracellular accumulation (b, d, e, g, j). The results of comparative analysis are marked in the graphs; significant differences were indicated by asterisks (* p‐value < 0.05; p‐value < 0.01; * p‐value < 0.001, and indicates p < 0.0001), while non‐significant differences were denoted as ns.

Excitation spectra of Ca10‐xDyx(PO4)6F2 recorded at room temperature, λem = 574 nm.

The temperature‐dependent emission spectra, λexc = 351 nm (a); luminescence intensity ratio LIR parameters (b); and relative sensitivity SR (c).

The temperature‐dependent excitation spectra, λem = 574 nm (a); luminescence intensity ratio LIR parameters (b); and relative sensitivity SR (c)

The energy level diagram of Dy3+ ions illustrates excitation, emission, and energy transfer mechanisms.

Emission spectra of Ca10‐xDyx(PO4)6F2 recorded at room temperature, λexc = 351 nm (a); the linear fitting of the y = log(I/x), x = Dy3+ concentration (b).

Luminescence decay curves of Ca10‐xDyx(PO4)6F2 detected at room temperature (a) and of Ca9.7Dy0.3(PO4)6F2 as a function of various ambient temperature (b).

Tables

TABLE 1. The elements’ content, the average crystallite sizes calculated by the Debye‐Scherrer method (DDS), and the estimated crystallinity degree (Xc ) for the Dy3+ doped powder as a function of Dy3+ ion concentration.

Sample	Dy3+ [mol]	F− [mol]	Ca+Dy/pmol/mol	DDS [nm]	σDS	Xc [%]
1 mol% Dy3+	0.12	1.08	1.65	30.4	4.94	62.7
2 mol% Dy3+	0.22	1.26	1.70	23.2	3.12	40.0
3 mol% Dy3+	0.36	1.21	1.64	23.6	2.93	40.1
5 mol% Dy3+	0.57	1.32	1.67	15.6	2.01	—
7 mol% Dy3+	0.75	1.38	1.70	13.4	2.00	—

TABLE 2. Antimicrobial activity of Dy3+‐doped fluorapatite against reference strains and clinical isolates.

Microorganisms	Type of test sample–x Dy3+ (mol%)
	1	2	3	5	7
	Growth inhibition zone (mm)
Escherichia coli ATCC13706	0.0 ± 0.0	2.0 ± 0.0	5.0 ± 1.0	7.0 ± 2.0	8.0 ± 2.0
Escherichia coli – clinical isolate	0.0 ± 0.0	0.0 ± 0.0	4.0 ± 1.0	5.0 ± 1.0	7.0 ± 1.0
Pseudomonas aeruginosa ATCC27853	0.0 ± 0.0	3.0 ± 1.0	7.0 ± 1.0	9.0 ± 2.0	10.0 ± 2.0
Pseudomonas aeruginosa – clinical isolate	0.0 ± 0.0	0.0 ± 0.0	5.0 ± 1.0	8.0 ± 1.0	9.0 ± 1.0
Streptococcus mutans ATCC25175	0.0 ± 0.0	3.0 ± 1.0	6.0 ± 1.0	11.0 ± 2.0	13.0 ± 2.0
Streptococcus mutans – clinical isolate	0.0 ± 0.0	0.0 ± 0.0	4.0 ± 1.0	9.0 ± 1.0	9.0 ± 1.0
Staphylococcus aureus ATCC 6538	0.0 ± 0.0	3.0 ± 1.0	5.0 ± 2.0	7.0 ± 2.0	8.0 ± 2.0
Staphylococcus aureus – clinical isolate	0.0 ± 0.0	0.0 ± 0.0	4.0 ± 1.0	5.0 ± 1.0	7.0 ± 1.0
Staphylococcus epidermidis NCTC 11047	0.0 ± 0.0	3.0 ± 1.0	6.0 ± 2.0	6.0 ± 1.0	8.0 ± 2.0
Staphylococcus epidermidis ‐ clinical isolate	0.0 ± 0.0	0.0 ± 0.0	5.0 ± 1.0	5.0 ± 1.0	8.0 ± 2.0
Enterobacter aerogenes ATCC 51697	0.0 ± 0.0	4.0 ± 1.0	6.0 ± 1.0	7.0 ± 1.0	7.0 ± 1.0
Enterobacter aerogenes – clinical isolate	0.0 ± 0.0	2.0 ± 1.0	5.0 ± 1.0	6.0 ± 1.0	5.0 ± 1.0

TABLE 3. Influence of the material on indicator microorganisms (reference strains and clinical isolates) by the liquid culture method—concentration of the test sample = 100 mg/mL.

Microorganisms	Type of test sample–x Dy3+ (mol%)
	1	2	3	5	7
	Number of microorganisms (x103 CFU)
Escherichia coli ATCC13706	0.13→3.9	0.20→1.7	0.19→ nd	0.20→nd	0.20→nd
Escherichia coli–clinical isolate	0.10→8.0	0.12→3.3	0.14→ nd	0.19→ nd	0.19→ nd
Pseudomonas aeruginosa–ATCC27853	0.14→3.7	0.12→nd	0.11→ nd	0.12→ nd	0.10→ nd
Pseudomonas aeruginosa–clinical isolate	0.11→3.6	0.11→ nd	0.15→ nd	0.15→ nd	0.10→ nd
Streptococcus mutans ATCC25175	0.10→3.9	0.19→ nd	0.19→ nd	0.19→ nd	0.10→ nd
Streptococcus mutans–clinical isolate	0.13→8.0	0.11→ nd	0.14→ nd	0.14→ nd	0.10→ nd
Staphylococcus aureus ATCC 6538	0.20→1.7	0.13→8.0	0.16→ nd	0.10→ nd	0.10→ nd
Staphylococcus aureus–clinical isolate	0.12→3.3	0.20→1.7	0.14→ nd	0.10→ nd	0.15→ nd
Staphylococcus epidermidis NCTC 11047	0.12→0.58	0.12→3.3	0.14→ nd	0.15→ nd	0.15→ nd
Staphylococcus epidermidis–clinical isolate	0.11→0.53	0.12→0.58	0.11→ nd	0.19→ nd	0.19→ nd
Enterobacter aerogenes ATCC 51697	0.10→1.4	0.13→8.0	0.12→ nd	0.11→ nd	0.11→ nd
Enterobacter aerogenes–clinical isolate	0.19→3.3	0.20→1.7	0.11→ nd	0.20→nd	0.15→nd

Full text

Introduction

1

Nowadays, materials possessing the apatite structure, co‐doped with lanthanide ions, are widely applied as phosphors, biolabeling agents, laser hosts, chemical catalysts, and up‐conversion materials. This is due to the combination of advantageous structural properties of apatite materials with excellent luminescent properties of lanthanide ions (Ln^3+^) [1, 2]. The properties of these materials can be easily tailored through a wide range of cationic and anionic substitutions involving species with similar radii and charges. The apatite structure can be described as A_10_[MO_4_]6_X_2, where A represents a cation (usually Ca^2+^ or Sr^2+^), MO_4_ ^n−^ is an anionic group (such as (PO_4_)^3−^, (SiO_4_)^4−^, (CO_3_)^2−^, etc.), and X is represented by OH^−^, F^−^, Cl^−^, Br^−^, or O^2−^. Remarkably, such modifications can be achieved without altering the fundamental structural framework. One possible substitute is implementing Ln^3+^ instead of the appropriate cation in a cationic site. Lanthanide ions possess unique luminescent properties due to f‐f transitions [3].

The high cytocompatibility of Dy^3+^ ion‐doped magnetic and luminescence multifunctional nanocomposite, i.e., Fe_3_O_4_@SiO_2_@GdVO_4_:Dy^3+^, was previously shown using the human cervical cancer cell line, i.e., HeLa [4]. Moreover, Dy^3+^‐doped nanomaterials have significant potential for biomedical applications due to their targeted mechanism of action. For example, nanosized phosphor, LWO:1.5 mol% Dy^3^⁺, developed by Kumar et al., showed high cytocompatibility toward normal cells, i.e., fibroblast cell line WI‐38, and induced apoptosis toward breast cancer cell line, MCF‐7, demonstrating its potential for selective anticancer applications [5]. Moreover, Vasanthavel et al. [6] also showed that Gd^3^⁺ and Dy^3^⁺ dual‐doped ZrO_2_–SiO_2_ systems are cytocompatible, with dose‐dependent cytotoxicity. While higher concentrations (200 µg/mL) exhibited increased toxicity toward the osteosarcoma cell line, MG‐63 cells, the lower concentrations induced less than 20% cytotoxicity, indicating a relatively favorable biocompatibility profile at reduced doses. However, we haven't found any information on the influence of Dy^3+^ on progenitor cells, such as bone marrow stromal cells, which play a crucial role in tissue regeneration, differentiation, and maintaining cellular homeostasis. Understanding how Dy^3^⁺ ion‐doped materials interact with these cells is essential for evaluating their potential applications in biomedicine, particularly in bone tissue engineering and regenerative therapies.

To obtain thermal images of living organisms without disruption, luminescent sensors with thermally responsive spectral characteristics are a promising solution. Phosphors doped with Ln^3+^ ions (e.g., Eu^3+^, Tb^3+^, and Dy^3+^ ions) offer high efficiency, a prolonged operational lifespan, and eco‐friendly characteristics. Among the ions used for phosphor preparation, the Dy^3+^ ion stands out due to its distinctive characteristics, which are useful as optical temperature sensors. Dy^3+^ ions were studied in various host matrices [7, 8, 9], emphasizing the importance of matrix influences on luminescence properties (e.g., signal intensity). Research has also been conducted on apatite‐type hosts. For instance, Liu et al. proposed Dy‐doped oxysilicate apatite as a potential optical thermometer [10], and Oliveira et al. [11] investigated the temperature and photoluminescence dependence of Eu^3+^/Dy^3+^ doped apatite‐type silicates.

A multifunctional luminomagnetic agent for bioimaging can be created by doping Dy^3+^ into the apatite crystal matrix. As with phosphors, the use of apatite materials as a host matrix is even more beneficial in this case. Apatites are highly biocompatible and bioactive, which enables their applications in bioimaging, tissue engineering, and drug delivery systems. Doping apatites with Dy^3+^ ions results in biomaterials with tunable, photoluminescent, and magnetic properties [12, 13]. Additionally, its cytocompatibility makes it suitable as a potential contrast agent for magnetic resonance imaging (MRI) [14, 15, 16]. Sánchez Lafarga et al. [17] studied the biological activity of Dy‐doped hydroxyapatite, functionalized with glucuronic or folic acids. A rat model assessed the in vivo toxicity of Dy‐substituted hydroxyapatite synthesized via the co‐precipitation method. Functionalization of the materials significantly reduced oxidative stress markers, such as lipoperoxides and nitric oxide. Notably, Dy^3^⁺‐doped fluorapatites (Dy^3+^‐FAp) exhibited enhanced luminescent properties compared to hydroxyapatites, attributed to the absence of hydroxyl groups that typically induce luminescence quenching.

The Dy^3+^ ions' luminescence exhibits two dominant emission bands, namely a yellow band (∼575 nm) and a blue band (∼480 nm), corresponding to the hypersensitive transition ^4^F_9/2_ → ^6^H_13/2_ (ΔL = 2, ΔJ = 2) and the ^4^F_9/2_ → ^6^H_15/2_ transition, respectively [18, 19]. By adjusting the yellow‐to‐blue intensity ratio (Y/B), it is possible to tune the chromaticity coordinates of Dy^3+^‐doped phosphors across the yellow, blue, and white light regions. Furthermore, using an apatite‐type matrix to host Dy^3+^ ions offers promising prospects as a white‐emitting component for LEDs [20]. For example, Zhang et al. proposed Ca_9_La(PO_4_)5(GeO_4_)F_2_:Dy^3+^ phosphor, and Deng et al. obtained Ba_2_Y_3_(SiO_4_)_3_F:Dy^3+^ phosphor (both materials synthesized via a facile solid‐state reaction) [3]. Results showed that both phosphors exhibit satisfactory thermal properties and internal quantum efficiencies, introducing them as potential materials for lighting technology.

However, to the best of our knowledge, no biological studies have been conducted to determine the potential molecular and cellular mechanisms of action of Dy^3+^‐doped fluorapatite. This status represents a significant gap in the literature, as such studies could provide valuable insights into cytotoxicity and potential biomedical applications of newly obtained materials. In this research, we present the study of the structural, temperature‐dependent luminescence, antimicrobial, and biological activity of Dy^3+^‐doped fluorapatite structure‐type materials, followed by a comprehensive characterization of the material. Our prior investigation explored the behavior of Dy^3+^ ions in silicate fluorapatite [21]. By leveraging theoretical models and detailed calculations, we attribute luminescence quenching to multipole–multipole interactions between Dy^3^⁺ ions. Extending this to phosphate fluorapatite (presented paper), we verified that energy transfer occurs solely via multipole–multipole interactions. The presented multi‐faceted approach offers valuable insights into the potential of Dy‐doped fluorapatite as a multifunctional biomaterial.

Result and Discussion

2

Physicochemical Characterization

2.1

X‐Ray Powder Diffraction

2.1.1

The phase identification of heat‐treated samples was carried out using X‐ray powder diffraction. Figure 1 presents the X‐ray diffraction patterns of samples with different Dy3+ ion concentrations. A pure hexagonal structure (P6_3_/m space group) was detected in all samples of apatite crystals. A comparison was made between the measured patterns and the theoretical ones originating from the ICSD database. There were narrow lines that corresponded with apatite structure at 2θ 25.9° (002); 28.1° (012); 29.0° (210); 32.0° (211); 33.1° (300); 34.2° (202); 40.0° (310); 46.9° (222); 49.6° (210); 53.2° (004). Corresponding Miler indexes are given in brackets. The results are in strong agreement with previous studies [22, 23, 24, 25].

X‐ray diffraction pattern of Ca10‐xDyx(PO4)6F2, where x = 0.1–0.7, heat‐treated at 600°C.

The calcium ions in the apatite host lattice occupy two non‐equal crystallographic positions. In the close vicinity of the Ca(1) calcium site, there are nine atoms from the PO_4_ ^3−^ groups. Therefore, the irregular coordination polyhedron with formula CaO_9_ and symmetry *C_3_

• is created in the environment of Ca(1). A calcium ion in the Ca(2) position is surrounded by six oxygen atoms from PO_4_ ^3−^ and one oxygen atom from the hydroxyl group or fluorine ion in the case of fluorapatite. The atoms surrounding the Ca(2) position cause an irregular polyhedron with the formula CaO_6_OH (or CaO_6_F). Depending on the ionic radius, various ions can substitute calcium ions and be substituted into their positions. Because both dysprosium and calcium have congruent ionic radii, the replacement by dysprosium is possible (ionic radii of Dy^3+^: 1.083 Å at CN_9_ and 0.97 Å at CN_7_ and Ca^2+^: 1.18 Å at CN_9_ and 1.06 Å at CN_7_) [26]. The substitution of a trivalent dysprosium ion for a divalent calcium position may cause the charge imbalance. The possible charge compensation mechanism may include the creation of interstitial oxygen, negative, and positive charge vacancies. It was comprehensively described in our previous papers [24, 27].

The elemental composition of the investigated materials was checked by EDS analysis (see Figure S1). The calculated amounts of Dy^3+^ ions, as well as the fluorine content, are presented in Table 1 and are consistent with the theoretical values. The results indicate the presence of a single fluorine atom in the tested materials; however, given the limitations of EDS in fluorine analysis, these results primarily confirm its presence. The stoichiometric apatite structure is defined by a cation‐to‐anion molar ratio of 1.67. The calculated ratio for the synthesized materials agrees with this value, considering the acceptable experimental error.

TABLE 1: The elements’ content, the average crystallite sizes calculated by the Debye‐Scherrer method (DDS), and the estimated crystallinity degree (Xc ) for the Dy3+ doped powder as a function of Dy3+ ion concentration.

With increasing Dy^3+^ concentration in the tested host lattice, the full width at half maximum (FWHM) rises (see Figure 1). This phenomenon is related to the decrease in crystal parameters with increasing dopant concentration because the dysprosium ionic radii are smaller than the calcium ionic radii. On the other hand, it is also observed a small shift in the peak location to the smaller 2θ angles, visible at 25.9° (002). Introducing dopant ions into the host lattice, even in small quantities, induces defects and structural distortions, thereby altering the material's internal structure.

X‐ray patterns were used to estimate crystallite size and crystallinity. According to the Debye‐Scherrer formula (Equation 10 – section Methods), the average crystallite sizes were calculated (D_DS_), and the results are listed in Table 1. The results are also compared to the parameters calculated by the Rietveld (*D_R_ *) method. All results are listed in Table S1. A decrease in crystallite size can be attributed to the increase in Dy^3+^ ion concentration. This state is caused by the substitution of calcium atoms by Dy^3+^ ions, which possess a lower ionic radius than calcium atoms. The average size of nanocrystals doped with 1 mol% Dy^3+^ ions is equal to 30.4 nm (*D_DS_ *), 24.1 nm (*D_R_ *), and it decreases up to 13.4 nm (*D_DS_ *), 10.9 nm (*D_R_ *) in the case of material doped with 7 mol% Dy^3+^ ions. The degree of crystallinity becomes lower as the dopant level is increased. The sample doped with 1 mol% Dy^3+^ ion is approximately 62.7%, and 40.1% for the sample containing 3 mol% Dy^3+^ ion. Finally, the crystallinity of samples doped with 5 and 7 mol% Dy^3+^ ions was too low to estimate the crystallinity degree with Equation 11 (see section Methods).

Microscopy Observation

2.1.2

The morphology of the 3 mol% Dy^3+^ ion‐doped sample was observed on the SEM images (see Figure S2—Supplementary Information). Aggregates approximately 1 µm in size are observed; however, individual nanosized spherical particles remain distinguishable. Diameter measurements are shown as a histogram in Figure S2c, indicating an average particle size between 60 and 100 nm.

Infrared Spectroscopy

2.1.3

The Fourier‐transformed infrared spectra (FT‐IR) and Raman spectra were recorded for the sample containing 3 mol% Dy^3+^ ions. Both spectra were displayed in Figure S3 (Supplementary Information). The samples were chosen because of their best luminescence performance. The FT‐IR spectra show the typical vibrational modes corresponding to the PO_4_ ^3−^ groups derived from the apatite matrix. The most intense line associated with asymmetric stretching *v_3_ * vibrations was found at 1034 and 1098 cm^−1^. The symmetric nondegenerate stretching *v_1_ * vibrations at 963 cm^−1^. As the matrix contains the F^−^ instead of OH^−^, we do not see the lines at about 633 cm^−1^, where the vibration of OH^−^ is detected in hydroxyapatite matrix [28]. The Raman shifts of the apatite matrix were located in the range of 400–1200 cm^−1^. In this range, the most intense band recorded at 965 cm^−1^ was associated with the symmetric stretching *v_1_

• of the P─O bond. The vibrational modes located at 428, 591, and 1053 cm^−1^ were attributed to the O─P─O linkage bending mode v 2 and v 4, and asymmetric stretching of the P─O bond v 3, respectively [29].

Microbiological Testing

2.2

Antibacterial Tests

2.2.1

The microbiological testing involved evaluating the antimicrobial properties of the Dy^3+^‐doped fluorapatite against six microbial species that pose an intraoperative microbiological threat, such as Escherichia coli, Pseudomonas aeruginosa, Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis, and Enterobacter aerogenes. The test used the well‐diffusion method, reference strains, and clinical isolates.

The antibacterial activity of Dy‐doped apatite was evaluated in terms of its ability to inhibit the expansion of bacterial colonies. The results of the growth inhibition area are presented in Table 2 and shown in Figure 2. The study includes testing against reference strains and clinical isolates. Across all the tested bacterial species, there is an increased zone of growth inhibition with increasing concentrations of Dy^3+^ ions. A sample containing 3 mol% of Dy^3+^ ions shows a positive influence on bacterial growth and inhibits it for approximately 3–4 mm. The highest inhibition is observed for the samples with the highest Dy^3+^ concentration of the Pseudomonas aeruginosa ATCC27853 (10 ± 2 mm), and Streptococcus mutans ATCC25175 (13 ± 2 mm).

The growth inhibition zone was tested for a Dy3+‐doped apatite. RS – reference stain, CI – clinical isolate.

Microbiological Activity in Liquid Culture

2.2.2

The biomass of microorganisms was diluted in 0.9% NaCl so that the concentration of microorganisms was 1.0 × 10^2^ cfu/mL. In parallel, a 100 mg/mL solution of each of the tested materials was prepared (the solvent was 0.9% NaCl). The prepared suspension of microorganisms was then added to the dilutions created in this manner for inoculation. The samples were combined and incubated for 18 h, at 37°C using media designed for a specific group of microorganisms. The quantity of bacteria was counted both before and after incubation.

Table 3 lists the quantity of each of the tested bacterial species before and after incubation. According to the results, increasing Dy^3+^ concentration affects the viability of microorganisms. In the case of Pseudomonas aeruginosa and Streptococcus mutans (reference strains as well as clinical isolates), the concentration of Dy^3+^ ions equal to 2 mol% was strong enough to reduce the bacterial population completely. With a Dy^3+^ concentration set at 3 mol%, the population of the rest of the microorganisms was reduced.

Our study confirms a reduction in the bacterial activity in the presence of Dy^3+^‐doped fluorapatite structure‐type nanosized crystals. The mechanism underlying the antibacterial activity of rare earth (RE) ions remains inadequately explored. Current evidence suggests that the antibacterial effect may be primarily associated with the adsorption of RE ions onto bacterial cell walls. This interaction is believed to be facilitated by the presence of multiple phosphate groups on the bacterial surface, which serve as binding sites for the RE ions. However, there is a notable lack of information regarding the potential penetration of RE ions through bacterial cell walls and their intracellular effects. Therefore, further and deeper research is needed into the mechanism of interaction between RE ions and microorganisms [30, 31, 32].

Biological Potential

2.3

Determination of Biomaterials Influence on Cellular Metabolism–Screening Assay

2.3.1

The metabolism of human bone marrow mesenchymal stromal cells (hBMSCs) in contact with Dy^3+−^doped nanosized apatite‐type materials was monitored with Alamar Blue dye, and measured after 72 h of culture. Furthermore, the morphology of hBMSCs was assessed through cytoskeleton and nuclei staining and analyzed using an epifluorescent microscope. This also enabled the evaluation of the internalization of luminescent biomaterials.

Results from the metabolic assay revealed that functionalized nanosized apatite‐type materials exhibited no cytotoxic effects across all tested concentrations, i.e., 0.5, 1, 10, 100, and 1000 µg/mL–indicating their high cytocompatibility (Figure 3a–e) and confirmed previous and confirms previous scientific reports on the low toxicity of Dy^3^⁺‐doped nanocomposites [6]. We observed either an enhancement in cellular metabolism in the presence of nanomaterials or no significant changes in metabolic activity, indicating their excellent cytocompatibility profile. The metabolic factor measured for hBMSCs cultured with Dy^3^⁺ ion‐doped nanomaterials at a concentration of 1 µg/mL demonstrated a significant enhancement in cell activity. This effect was dose‐dependent, particularly for nanocrystals functionalized with 1, 2, and 3 mol% Dy^3^⁺ (Figure 3a, b, d).

*The effect of Dy3⁺ion‐doped nanosized apatite‐type materials on the metabolism of human bone marrow stromal cells (hBMSCs). Cellular metabolism was assessed using the Alamar Blue assay, with results expressed as the metabolic factor relative to the activity of cells in the control cultures (expressed as 1). A comparative analysis was conducted using results from four independent experiments. The data are displayed as bar graphs, representing the mean ± standard deviation (SD). Statistical significance is denoted by asterisks, where indicates p‐value  < 0.05; ** p‐value < 0.01; *** p‐value < 0.001, and **** indicates p‐value < 0.0001, while “ns” denotes non‐significant differences.

The fact that nanomaterials maintained a high level of cytocompatibility across a broad concentration range was also evidenced by the proper morphology and growth pattern of hBMSC observed in cultures with biomaterials’ presence. Additionally, the nanosized biomaterials were efficiently internalized by the cells, but their intracellular distribution did not exhibit a clear dose‐dependent relationship (Figure 4).

The effect of Dy3⁺ion‐doped nanosized apatite‐type materials on the morphology and growth pattern of human bone marrow stromal cells (hBMSCs). Epifluorescence microscopy was used to monitor potential changes in hBMSCs culture morphology resulting from biomaterial exposure. The nuclei were stained with DAPI (blue signal), while the actin cytoskeleton was counterstained with phalloidin Atto‐488 (green signal). The red signal corresponds to the nanomaterials doped with Dy3⁺ ions. The cells were visualized at 100‐fold magnification. The scale bar (see in control cultures) equals 200 µm.

Since all Dy^3^⁺ion‐doped nanocomposites introduced to hBMSC cultures at a concentration of 1 µg/mL were observed to support cell metabolism. We selected this concentration for further, more detailed assays to investigate their effects on hBMSC cellular health. This concentration was determined to be optimal, as it promoted cellular activity without exhibiting any cytotoxic activity.

Evaluation of Dy3⁺ Ion‐Doped Nanosized Apatite‐Type Materials on Cellular Health–In‐Depth Analysis of Viability, Mitochondrial Function, and Molecular Markers Modulated by Nanomaterials

2.3.2

In order to determine the effect of nanocomposites on cellular health, we performed a detailed evaluation of their impact on cell death profiles. The analysis demonstrated that the viability of cells can be modulated by Dy^3+^ ion‐doped nanosized apatites. Specifically, nanosized apatites doped with 2 mol% and 5 mol% of Dy^3+^, when applied at a concentration of 1 µg/mL, significantly enhanced the viability of human bone marrow‐derived mesenchymal stem cells (hBMSCs).

For the remaining nanocomposites, no significant impact on cell viability was observed, as the percentage of live cells remained comparable to that in the control cultures. Importantly, in reference to the control group (untreated cells), all tested biomaterials effectively reduced the percentage of early and late apoptotic hBMSCs, indicating a potential anti‐apoptotic effect, confirming also observations of Vasanthavel et al. on MG63 cells [6].

However, a notable increase in the percentage of necrotic cells was observed in cultures treated with nanocomposites, with the exception of those doped with 5 mol% Dy^3+^ ions. The Dy^3+^ ions may compromise cell membrane integrity and thus induce loss of cellular homeostasis, leading to necrosis, while reducing the number of apoptotic cells (see Figure 5). Those findings indicate the potential dual effect of Dy^3^⁺ ions on cell fate. This partially corresponds with the data from Kumar et al., showing that its biological impact is a complex and probably multi‐level process also regulated by the concentration of Dy^3+^ ions [5].

The influence of Dy3+‐ doped nanocomposites on the viability of human bone marrow stromal cells (hBMSCs). The representative images (panel a) show the distribution of cells based on Annexin FITC‐A staining, allowing distinguishing of different cell populations: live cells (LL—lower left quadrant), early apoptotic cells (LR—lower right quadrant), late apoptotic cells (UL—upper left quadrant), and necrotic cells (UR—upper right quadrant) (a). A comparative analysis of apoptotic cell populations was conducted (b‐d), with significant differences indicated by asterisks (** p < 0.001; ** p < 0.01; * p < 0.05), while nonsignificant differences are denoted as ns (b).*

To obtain information on potential molecular pathways, we analyzed the expression of several transcripts associated with apoptosis (Figure 6). We observed a tendency toward reduced expression of BAX and BAD in hBMSCs cultured with Dy^3+^ ion‐doped nanomaterials, which may suggest a decrease in pro‐apoptotic signaling (Figure 6a,b). However, at the same time, anti‐apoptotic BCL2 mRNA expression was also downregulated in groups treated with Dy^3^⁺‐doped nanocomposites (Figure 6c).

The influence of Dy3+ ion‐doped nanosized apatite‐type materials on the expression of key apoptosis‐ and cell cycle‐regulating genes. The analysis included the determination of pro‐apoptotic markers, i.e., BAD and BAX, and anti‐apoptotic markers, i.e., BCL‐2 (c) and BCL‐XL (g). Moreover, the BAX/BCL‐2 ratio was determined to assess the balance between pro‐ and anti‐apoptotic signaling (d). Additionally, mRNA levels of p53 (e) and p21 (f) were analyzed to evaluate the activation of cell cycle regulatory pathways. A comparative analysis was conducted to evaluate the significance of the observed expression patterns. Statistically significant differences were indicated as follows: * p‐value < 0.05; ** p‐value < 0.01; *** p‐value < 0.001, and **** indicates p < 0.0001, while nonsignificant differences are denoted as ns (b).

To better assess the overall impact of the Dy^3+^ ions on apoptosis regulation, we calculated the BAX/BCL2 ratio, a key marker of apoptotic susceptibility [33], to determine whether the balance between pro‐ and anti‐apoptotic signaling favored cell survival or apoptosis (Figure 6d). This ratio was markedly increased in cells treated with Dy^3+^‐doped nanomaterials at concentrations of 2, 3, 5, and 7 mol%, indicating a shift toward a pro‐apoptotic state at higher Dy^3^⁺ ion concentrations.

Moreover, the Dy^3+^ ion‐doped composites modulated the expression of p53 [34], a critical regulator of apoptosis and cellular stress responses. In hBMSCs, treated with nanocomposites doped with 3 and 7 mol% Dy^3^⁺ ions, we observed significantly increased mRNA levels of p53, mirrored by the upregulation of p21 mRNA levels (Figure 6e, f). This may indicate an activation of the p53/p21 axis, a pathway commonly associated with cell cycle regulation and stress response [34, 35]. We also noted the downregulation of BCL‐XL, which may suggest a potential compromise in mitochondrial integrity and a shift in the regulation of apoptotic pathways toward a BCL‐2‐dominated survival mechanism (Figure 6g). This imbalance could render cells more susceptible to mitochondrial stress and non‐apoptotic forms of cell death, such as necrosis, which was noted. Thus, we decided to analyze the influence of the obtained Dy^3+^ ion‐doped nanomaterials on mitochondrial metabolism.

The analysis of mitochondrial membrane potential (Δ*Ψ_m_ ) revealed a concentration‐dependent effect of Dy^3+^ ion‐doped nanosized apatite‐type materials on mitochondrial function (Figure 7a–c) [36]. In the untreated group, a high percentage of cells displayed elevated ΔΨ_m_ , indicative of healthy mitochondrial polarization. Nanosized materials functionalized with Dy^3+^ ions at concentrations of 1 and 2 mol% preserved mitochondrial function (Figure 7b), as evidenced by a similar percentage of cells with high ΔΨ_m_

• compared to the untreated control. However, higher Dy^3+^ concentrations (3, 5, and 7 mol%) significantly increased the proportion of cells with lowered Δ*Ψ_m_ *, indicative of mitochondrial depolarization and dysfunction (Figure 7c). Obtained findings emphasize the importance of optimizing Dy^3+^ ion‐doping levels to balance nanomaterials' cytocompatibility and minimize mitochondrial toxicity in biomedical applications. Thus, we decided to check the expression of mitochondrial homeostasis and quality control key regulators (Figure 8).

The influence of Dy3+‐ doped nanocomposites on mitochondrial membrane potential (ΔΨm ) of human bone marrow stromal cells (hBMSCs). The representative images (panel a) show the distribution of cells based on the fluorescence intensity of JC‐1 dye, indicating changes in ΔΨm . Cells with high ΔΨm exhibit red fluorescence due to J‐aggregates, whereas cells with depolarized mitochondria show green fluorescence from JC‐1 monomers.(a). A comparative analysis of cells with high and low ΔΨm was performed (b‐c), with significant differences indicated by asterisks ( p‐value  < 0.05; *** p‐value < 0.001 and **** indicates p < 0.0001) while nonsignificant differences are denoted as ns.*

The influence of Dy3+‐doped nanocomposites on the expression of regulators of mitochondrial dynamics, mitophagy, and mitochondrial quality control. The analysis included mRNA expression of MFN1 (a), PINK1 (c), PARKIN (f), FIS1 (h), and miR‐19a‐3p (i). Protein‐level assessment involved MFN1, PINK1, and PARKIN abundance and intracellular accumulation (b, d, e, g, j). The results of comparative analysis are marked in the graphs; significant differences were indicated by asterisks ( p‐value < 0.05; ** p‐value < 0.01; *** p‐value < 0.001, and **** indicates p < 0.0001), while non‐significant differences were denoted as ns.*

The expression of MFN1 (mitofusin 1), a key regulator of mitochondrial fusion [37], was significantly upregulated at the mRNA level in cells treated with Dy^3+^‐doped nanomaterials, with the exception of the 2 mol% Dy^3^⁺ group, where MFN1 mRNA levels were comparable to those of the untreated group (Figure 8a). However, Western blot analysis showed no significant changes in MFN1 protein levels across all culture conditions, suggesting intracellular stability and the preservation of mitochondrial network dynamics, as well as the function of hBMSCs exposed to Dy^3+^ion‐doped nanocomposites (Figure 8f, j) [37, 38].

Simultaneously, the expression of FIS1 (fission protein), associated with the regulation of mitochondrial fission [39], was modulated by Dy^3+^ ion‐doped nanosized materials. The mRNA levels for FIS1 were significantly upregulated when Dy^3+^ ion was doped into nanomaterials at concentrations of 2, 5, and 7 mol%. In comparison, no significant changes in FIS1 expression were observed in groups treated with 1 and 3 mol% of Dy^3+^ ions. The obtained data suggested that Dy^3+^ ion‐doped nanomaterials may induce mitochondrial remodeling. Moreover, increased mitochondrial fission may be triggered at higher Dy^3+^ ion concentrations, as indicated by the upregulation of FIS1 mRNA (Figure 8b). The data confirm previous observations, demonstrating that high concentrations of Dy^3^⁺ ions may reduce the mitochondrial activity of hBMSCs (Figure 7). Moreover, all nanocomposites doped with Dy^3+^ ions caused a significant reduction in miR‐19a‐3p levels in hBMSCs, suggesting a potential regulatory role of this microRNA in maintaining cellular homeostasis, particularly under conditions of Dy^3+^ ion exposure (Figure 8e). Given that miR‐19a‐3p is involved in the regulation of apoptosis, proliferation, and stress response pathways [40, 41], its downregulation may reflect an adaptive or stress‐induced response triggered by higher Dy^3+^ ion concentration.

Moreover, the protein levels of PINK1 and PARKIN, responsible for mitochondrial quality control and the regulation of mitophagy [42], remained unchanged across all Dy^3+^ ion‐doped material treatment groups (Figure 8g–j). In turn, mRNA levels for PINK1 were elevated in all groups except those treated with 3 and 5 mol% Dy^3+^, where no significant changes were observed (Figure 8c). In addition, PARKIN mRNA levels increased at 5 mol% Dy^3^⁺ ions but remained unaltered in the other groups (Figure 8d). The observed expression profile of PINK1 and PARKIN suggests a specific regulation of mitophagy‐related genes and the activation of mitochondrial quality control pathways in hBMSCs treated with Dy^3+^‐doped nanomaterials. This modulation may reflect an adaptive cellular response to maintain mitochondrial integrity and homeostasis under Dy^3+^ ion‐induced stress.

Luminescence Properties

2.4

Excitation Spectra

2.4.1

The excitation spectra measured for Dy^3+^:FAp powders monitored at emission wavelength 574 nm are presented in Figure 9. The spectra consist the series of excitation peaks, corresponded with the transitions from the ground state * ^6^H_15/2_ * to the excited states * ^4^F_3/2_

• (256 nm); *(^4^K, ^4^L)13/2
• (295; 298 nm); * ^4^M_17/2_
• (325.5 nm); * ^6^P_7/2_
• (351 nm); * ^6^P_5/2_
• (365 nm); * ^4^I_13/2_
• (389 nm); * ^4^G_11/2_
• (425 nm); * ^4^I_15/2_
• (451.5 nm). The energy diagram illustrates the pumping transitions (see Figure 12). The most intense luminescence is observed in the case of 3 mol% Dy^3+^ ion‐doped samples. Therefore, a detailed temperature‐dependent study was conducted for this sample.

Excitation spectra of Ca10‐xDyx(PO4)6F2 recorded at room temperature, λem = 574 nm.

Temperature‐Dependent Luminescence Properties

2.4.2

It was evaluated whether dysprosium‐activated fluorapatite is thermosensitive by examining its temperature‐dependent luminescence properties. The temperature‐dependent emission spectra were recorded under the excitation wavelength of 351 nm to populate the * ^6^P_7/2_

• excitation level. The excitation spectra were observed at 575.5 nm, the most intense emission line. The ambient temperature range was 80–600 K for emission and excitation spectra.

On the spectra, typical lines corresponding to the emission are detected because of the non‐radiative transfer of energy to the first excitation level * ^4^F_9/2_ *. It is observed energy transfers: * ^4^F_9/2_→^6^H_15/2_

• (479.5 nm); * ^4^F_9/2_→^6^H_13/2_
• (574.0 nm); * ^4^F_9/2_→^6^H_11/2_
• (662.5 nm); * ^4^F_9/2_→^6^H_9/2_
• (753.5 nm). The spectra are presented in Figure 10. Low temperature provides a possibility to observe emission from the excited level to the multiplet ground levels. The spectra recorded at 80 K (liquid nitrogen) present a series of narrow, sharp lines. A common application of this phenomenon in spectroscopy is to investigate atomic and molecular energy levels. The cooling of a sample to a low temperature allows scientists to isolate and study specific transitions between energy levels that would otherwise be less pronounced at higher temperatures. Otherwise, on the spectra recorded at higher temperatures, the radiation transition from higher excitation levels: * ^4^I_15/2_ → ^6^H_15/2_
• (455 nm) and * ^4^I_15/2_ → ^6^H_13/2_
• (539 nm) is visible. It should be pointed out that the luminescence intensity corresponded with the transition from the * ^4^F_9/2_ *, which decreased rapidly with temperature increase, and the transition from the * ^4^I_15/2_
• started to increase with ambient temperature. At higher temperatures, the thermal energy of the system is greater, resulting in a pumping of higher energy states, and the emission intensity from excited levels increases. For this purpose, emission spectra were quantitatively analyzed to evaluate the thermosensitivity of dysprosium‐activated materials.

The temperature‐dependent emission spectra, λexc = 351 nm (a); luminescence intensity ratio LIR parameters (b); and relative sensitivity SR (c).

The excitation spectra recorded as a function of various ambient temperatures show a series of pumping lines associated with the f‐f transition. There have been detected transitions between the ground state ^6^H_15/2_ and the excited states similar to what was observed in the room‐temperature measurements (see Figure 11). A series of tapering lines emerges as the ambient temperature decreases. As the temperature decreases up to 80 K, the most intense line appears at 347.5 nm, rather than 351 nm, which was most intense at room temperature and at higher temperatures.

The temperature‐dependent excitation spectra, λem = 574 nm (a); luminescence intensity ratio LIR parameters (b); and relative sensitivity SR (c)

The luminescence intensity ratio (LIR) and relative sensitivity (*S_R_ *) are the temperature‐dependent parameters. The LIR is calculated as a ratio between the integrated intensity of a given part of the spectrum and Equation 1. The *S_R_

• is performed to quantify the observed changes, estimated by Equation 2. The method can be applied to the assessment of material temperature sensing [43, 44, 45, 46].

$$[eqn]$$ $$[eqn]$$

For the presented study, three pairs of emission lines were chosen. According to certain ranges, the LIR parameters were calculated by the following Equations (3), (4), and 5:

$$[eqn]$$ $$[eqn]$$ $$[eqn]$$

In the case of temperature‐dependent excitation spectra [47], the two ranges were used for calculations (Equations 6 and 7):

$$[eqn]$$ $$[eqn]$$

Figures 10a and 11a present the temperature‐dependent emission and excitation spectra, measured in the ambient temperature range of 80–600 K. Based on the recorded spectra, LIR parameters were calculated (see Figures 10b and 11b). To achieve a high relative sensitivity (S_R_) parameter, the presented range was chosen. Furthermore, the proposed solution compares the wide and narrow ranges of the emission lines, and the emission corresponds to the various f‐f transitions. A general rule of thumb for selecting a spectral range is to compare a line that decreases with temperature with its opposite line that increases. In the case of excitation spectra, there were two ranges used for LIR calculation. The *LIR_EXC1_

• used the line that decreases rapidly with increasing temperature, and the relatively stable line. The *LIR_EXC2_
• parameter compares the two most common laser wavelengths, 351 and 405 nm. The luminescence intensity ratio (LIR) is enhanced as the temperature increases for all tested spectral ranges. The highest LIR value was obtained by comparing the excitation transition * ^6^H_15/2_→^6^I_15/2_
• (453–455 nm) with the * ^6^H_15/2_→^6^P_7/2_
• (348–353 nm).

The relative sensitivity (*S_R_ *) parameter value depends on the ambient temperature. The S_R_ parameters calculated based on the emission spectra are weak in the range of low temperatures (see Figure 10c). They start to increase when the temperature is higher than 220 K. The maximum is observed at 360 K, and it is equal to 0.67%/K (*LIR_EM1_ *). Up to 560 K, the *S_R_

• parameter is higher than 0.5%/K. A significantly different trend was observed for S_R_ parameters estimated from excitation spectra (see Figure 11c). The *S_R_
• is highest at low temperature (80 K), and decreases gradually as the temperature increases. Furthermore, both *S_R_
• values (*S_R EXC1_
• and *S_R EXC2_ *) are close to 0.5%/K. The combination of those two proposed calculations yields a temperature sensor with relatively stable sensitivity in the wide ambient temperature range. This leads to the conclusion that the proposed material doped with 3 mol% of Dy^3+^ ions may work efficiently equipped with two very common excitation laser diodes (351 and 405 nm) and narrow emission detectors (440–490 nm).

The investigated luminescence properties allow for the proposal of the energy level diagram. Figure 12 presents the pumping channels (excitation wavelength), observed depopulation channels (emission), possible cross‐relaxation energy transfers, and the transitions chosen for the *LIR_EXC2_

• and *LIR_EM1_
• calculations.

The energy level diagram of Dy3+ ions illustrates excitation, emission, and energy transfer mechanisms.

Concentration Quenching

2.4.3

The emission spectra of Dy^3+^ ion‐doped fluorapatite recorded at room temperature are shown in Figure 13a. The emission from the ^4^F_9/2_ excited state to the ground states was detected as a series of narrow lines * ^6^H_15/2_

• (480 nm); * ^6^H_13/2_
• (574 nm); * ^6^H_11/2_
• (662.5 nm); * ^6^H_9/2_
• (750.5 nm). The luminescence intensity increases with the Dy^3+^ ion concentration up to 3 mol% and rapidly decreases with further increases of Dy^3+^ ion concentration. There is a phenomenon known as concentration quenching that frequently occurs in rare‐earth element luminescence. Energy is transferred between closely located luminescent ions with corresponding distances between energy levels through a non‐radiative process. Energy may be transferred through a few non‐radiative channels. The depopulation of the * ^4^F_9/2_
• level may occur as a resonant energy transfer between * ^4^F_9/2_ → ^6^H_15/2_
• ↔ ^6^H_15/2_ → ^4^F_9/2_ (RET≈20,800 cm^−1^); and as a cross‐relaxation (CR) between * ^4^F_9/2_ ^6^H_9/2_ ↔ ^6^H_15/2_ → ^6^F_3/2_
• (CR1≈13,300 cm^−1^); * ^4^F_9/2_ → ^6^H_7/2_ ↔ ^6^H_15/2_ → ^4^F_5/2_
• (CR2≈12 800 cm^−1^); * ^4^F_9/2_ → ^6^F_1/2_ ↔ ^6^H_15/2_ → ^6^H_9/2_
• (CR3≈9,500 cm^−1^) [48]. The possible cross‐relaxation canals are marked in the energy diagram in Figure 12.

Emission spectra of Ca10‐xDyx(PO4)6F2 recorded at room temperature, λexc = 351 nm (a); the linear fitting of the y = log(I/x), x = Dy3+ concentration (b).

To determine the critical distance between dysprosium ions that produces the most efficient photoluminescence, we used the emission spectra of the samples with 3 mol% concentration. According to the theory described in a previous paper [21], we performed the detailed analysis, see Supporting Information.

According to our estimates, the critical radius is 10.77 Å. With a distance of more than 5 Å between two optically active centers, only a multipole‐multipole interaction is possible. The exchange interaction becomes more effective when the distance is reduced. Accordingly, multipolar interactions are considered to be the mechanism involved in the effective photoluminescence of Dy^3+^ ions in the investigated apatite host lattice. By looking at the linear correlation between the intensity of the photoluminescence and the concentration of Dy^3+^ ions, we analyzed the electrical multipolar character of the luminescence (more in the Supporting Information).

The proposed calculation suggests that the most dominant is the dipole‐dipole energy transfer. The nature of resonance energy transfer in various Dy^3+^ ion‐doped materials has previously been investigated [21, 49, 50, 51]. There is a theoretical explanation for concentration quenching in solid systems. In this model, excitation energy migrates from one activator center to another, causing quenching to be a non‐radiative process.

Comparison with the Silica‐Substituted Matrix

2.4.4

In the previous study, we investigated the silica‐substituted fluorapatite doped with the Dy^3+^ ions in the range of 0.5–5 mol%. The chemical formula of the investigated series of samples is Ca_10‐x_Dy_x_(PO_4_)2(SiO_4_)4_F_2 (here Si‐FAp:Dy), where x = 0.05; 0.1; 0.2; 0.5, where the current study discusses the phosphore‐fluorapatie of the general chemical formula: Ca_10‐x_Dy_x_(PO_4_)6_F_2 (here P‐FAp:Dy), where x = 0.1; 0.2; 0.3; 0.5; 0.7. The ongoing investigation also involved a comparison with a previous study to examine the matrix composition, focusing especially on the presence of silica within the crystal structure and its impact on luminescence characteristics.

Crystal structure analysis indicated that phosphate fluorapatite possesses higher structural stability compared with silica fluorapatite. The matrix without silica proved more capable of accommodating increased Dy^3^⁺ ion concentrations up to 7 mol%, whereas the silica fluorapatite showed phase separation, with 10% (w/w) DyPO_4_ detected at just 5 mol% Dy^3^⁺ incorporation.

Although the previous paper describes silica‐substituted fluorapatite with a critical distance between optically active ions very similar to that observed in the present study (10.79 vs. 10.77 Å), concentration quenching occurs at 1 mol% Dy^3^⁺ ions in the silica‐substituted material, compared to 3 mol% in the current case. The ability to incorporate a significantly higher concentration of Dy^3^⁺ ions, while still maintaining strong luminescence, may be attributed to several factors.

First, silica substitution induces distortions in the local symmetry, which can promote non‐radiative energy transfer. Second, silica substitution leads to a charge imbalance, which the structure may compensate for through crystal lattice distortions. These distortions can create non‐radiative relaxation centers. According to Kröger–Vink notation, charge compensation may involve the introduction of interstitial oxygen, OH^−^ ions replacing F^−^, or the formation of vacancies at O, Ca, or OH^−^ lattice sites, all of which can affect luminescence efficiency [27, 52]. It is also worth noting that silica‐containing matrices, in contrast to pure phosphate hosts, typically have a higher phonon energy. As a result, multi‐phonon relaxation occurs, which further enhances non‐radiative decay.

Lifetimes

2.4.5

In this study, decay profiles for the ^4^F_9/2_→^6^H_13/2_ transition were analyzed at room temperature for a variety of Dy^3+^ doping concentrations (Figure 14a) and as a function of ambient temperature for a 3 mol% Dy^3+^‐ doped sample (Figure 14b). The Dy^3+^ ions are located in two unequal crystallographic positions within the host lattice of apatite. It occurs with nonlinear luminescence kinetics. The PL kinetics curves were fitted using the exponential equation (see equation 8). Fitting curves are shown for the extremes of the tested series. This phenomenon comes with a nonlinear property, which led to the estimation of the average lifetime (*τ_AV_ *) using Equation 9.

$$[eqn]$$ $$[eqn]$$

Luminescence decay curves of Ca10‐xDyx(PO4)6F2 detected at room temperature (a) and of Ca9.7Dy0.3(PO4)6F2 as a function of various ambient temperature (b).

Where:

τAV – average lifetime

τ1 and τ2 – exponential lifetimes

A1 and A2 – nonlinear fitted parameters

Figure 14a presents the luminescence profiles of the material with different Dy^3+^ ion concentrations. Slight shortening of average lifetimes is observed with increasing dopant ion concentration, from 1.04 ms (1 mol% Dy^3+^ ions) to 0.64 ms (7 mol% Dy^3+^ ions). Luminescent materials are characterized by energy transfer processes between dopant ions. Increasing the concentration of optically active ions promotes an increase in the probability of energy transfer because the distance between them is smaller. A greater amount of energy can therefore be transferred from excited ions to neighboring ions, leading to a faster luminescence rate. On the other hand, in an environment with a higher concentration of optically active ions, nonradiative concentration quenching is more probable. This proximity can also promote non‐radiative energy transfer to lattice defects or quenching centers, which bypass PL emission [53]. Lifetimes measured at various ambient temperatures show the stable luminescence kinetics in the range of 80 to 600 K. The average lifetimes are in the range of 1.0 to 0.96 ms (see Table S3).

Conclusions

3

This study explores the unique combination of luminescence‐based sensing, biocompatibility, and antimicrobial properties of Dy^3+^ ions in nanosized fluorapatite, making them promising for advanced bioapplications. Fluorapatite nanoparticles, obtained through a wet‐chemistry synthesis process and crystallized in a hexagonal (P6_3_/m) structure, demonstrate an elemental composition in agreement with the initial assumptions. We have examined the structural, luminescence, antimicrobial, and biological properties of Dy^3+^ ion‐doped fluorapatite‐type materials.

The Dy^3+^‐doped nanocomposites demonstrate antimicrobial potential, suggesting their possible role in infection prevention. Their low cytotoxicity toward human bone marrow‐derived progenitor cells strongly supports their cytocompatibility. The biological features of the obtained nanomaterials make them promising candidates for further exploration in biomedical applications.

The photoluminescence characterization was studied based on the excitation, emission spectra, and luminescence kinetics. Several wavelength ranges were selected for the temperature‐dependent analysis. By LIR parameter calculations, we can obtain a temperature sensor with a relatively stable sensitivity over an expansive range of ambient temperatures. Therefore, the proposed material doped with 3 mol% Dy^3+^ ions is likely to perform well when equipped with two very common laser diodes for excitation (351 and 405 nm) and narrow emission detectors (440–490 nm).

Materials and Methods

4

Synthesis

4.1

The series of Dy^3+^ ion‐doped fluorapatite‐type nanosized materials was synthesized by the co‐precipitation wet process. The starting products were: Ca(NO_3_)2 ∙4H_2_O (99.0‐103.0% Alfa Aesar), (NH_4_)2_HPO_4 (>99.0% Acros Organics), Dy_2_O_3_ (99.9% Ubichem), NH_4_F (>98.0%, Alfa Aesar). List of samples with the general formula: Ca_10‐x_Dy_x_(PO_4_)6_F_2, where x = 0.1; 0.2; 0.3; 0.5; 0.7. The concentration of Dy^3+^ ions was set as a ratio of calcium ion molar content. The first step of synthesis was recrystallization of Dy_2_O_3_ with HNO_3_ (65% suprapure Merck) to obtain the water‐soluble dysprosium(III) nitrate. Then, the rest of the substrates were dissolved in deionized water separately. All the substrates were used in stoichiometric amounts to obtain 2 g of final product. Substances dissolved in Teflon were mixed. A pH of 10 was adjusted by adding ammonia solution (NH_3_∙H_2_O 25% Avantor, Poland). Hydrothermal synthesis was conducted for 90 min at 240°C and autogenous pressure (40–45 bar) by the microwave reactor (ERTEC MV 02‐02). Obtained powders were cleaned and dried before being heat‐treated at 600°C for 3 h.

Physicochemical Characterization

4.2

Diffraction of X‐ray powders was used to determine the crystal structure and phase purity. The X‐ray diffraction powder patterns (XRPD) measured by the PANalytical X'Pert Pro X‐ray diffractometer equipped with Ni‐filtered Cu Kα_1_ radiation (Kα_1_ = 1.54060 Å, U = 40 kV, I = 30 mA) were analyzed by software Match! 3.7.0.124 version. The X‐ray patterns were used to estimate the crystallite size and the degree of crystallinity. The average crystallite sizes (D) were calculated using the Debye‐Scherrer formula (Equation 10):

$$[eqn]$$

where D is the crystallite size, K means the Scherrer constant (0.89), λ is the X‐ray wavelength (0.15406 nm), β refers to the FWHM (°) of the three selected reflections, (002), (222), and (213); and its θ angle in cos θ.

The crystallinity degree was estimated using the formula (Equation 11):

$$[eqn]$$

where I_300_ is the intensity of (300) reflection, and *V_112/300_

• is the intensity of the valley between (112) and (300) reflections.

In order to perform the elements' concentration, EDS equipment was used. Powders were tested by FEI Nova NanoSEM 230 scanning electron microscopy equipped with an EDS spectrometer (EDAX GenesisXM4). The operating acceleration voltage was set in the range 3.0–15.0 kV, and spots were set in the range 2.5–3.0. Equations 12 and 13 were used to calculate the concentrations of dopant ions:

$$[eqn]$$ $$[eqn]$$

Scanning Electron Microscopy

4.3

The SEM images were detected by Marca JEOL, model JSM‐IT500HR (Tokyo, Japan). For the measurement, the colloidal solution of the materials was prepared with isopropanol. The sample was coated with a 5 nm layer of carbon by using the Sample Sputter Coater SCD 050 (Bal‐Tec, Wallruf, Germany).

Fourier‐Transformed Infrared Spectra

4.4

The analysis was performed by the NICOLET IS5 spectrometer, iD1 Transmission–Thermo Scientific. The KBr pellet was prepared by hydraulic press, for 1 min, under 6 tons of pressure. The spectra were recorded in the range of wavenumbers 4000–400 cm^−1^, 32 scans, resolution 2 cm^−1^.

Raman Spectra

4.5

The Raman spectra were recorded by Micro Raman – Spectrometer Raman model Lab RAM HR, Horiba Jobin Yvon, equipped with the 633 nm laser. A small amount of sample was placed on the holder, and a scan was recorded with 30 s of acquisition time.

Microbiological Research–Methodology

4.6

Analysis of Dy‐doped nanosized apatite‐type materials microbiological purity. The study used the flooding method and nutrient agar medium with 2% glucose for total microbial counts and Sabouraud medium for mold and yeast counts. The incubation temperature for mesophilic microorganisms was 37°C and for psychrophiles was 18°C.

Antimicrobial Activity

4.7

Indicator microorganisms were transferred to test tubes containing Mueller–Hinton medium. They were cultured at 37°C for 24 h. Next, the liquefied agar medium was inoculated with 10% (v/v) 24 h indicator culture and poured into Petri dishes to obtain a distinct confluent layer. After solidification of the broth medium inoculated with the indicator microorganisms (Escherichia coli ATCC13706, Pseudomonas aeruginosa ATCC27853, Streptococcus mutans ATCC25175, Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis NCTC 11047, Enterobacter aerogenes ATCC 51697), wells were made with a cork borer. Each well was supplemented with 100 mg of Dy‐doped nanosized apatite‐type materials. Next, the diameters of the indicator bacteria growth inhibition or reduction were measured. The inhibition of the growth of the indicator microorganisms was manifested by complete lighting around the place where the hydrogel was transferred. It indicated the bactericidal activity of the bacterial strain. Bacteriostatic properties were determined by measuring the diameter of the growth inhibition zone (indicator strain growth limitation).

Cell Culture

4.8

Human bone marrow stromal cells (hBMSCs/SCC034), with established multipotency, were obtained from Merck Life Science (Poznan, Poland) and were purchased at passage 2 (p2). The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Warsaw, Poland) and with 1% of penicillin/streptomycin mix (P/S, Sigma–Aldrich/Merck, Poznan, Poland). The complete growth medium (CGM) was refreshed every 2–3 days to maintain optimal conditions. Cell cultures were maintained under aseptic conditions, and regular monitoring was performed to detect potential Mycoplasma contamination. The cells were incubated at 37°C, in a humidified atmosphere of 95% air and 5% CO_2_. Upon reaching 80% confluence, the cultures were passaged using a trypsin solution (StableCell Trypsin, Merck, Munich, Germany). The detailed protocol has been previously described [54]. The cells used for these experiments were at passage number 4 (p4).

Analysis of Cytotoxicity of Dy3+‐Doped Nanocomposites–Screening Test on Human Bone Marrow‐Derived Stromal Cells (hBMSCs)

4.9

To assess the influence of Dy^3+^‐doped nanomaterials on hBMSCs biology, cells were seeded at a density of 2 × 10^4^ cells per well in 24‐well plates pre‐coated with glass coverslips for subsequent microscopic analysis. The cells were cultured in 0.5 mL of complete growth medium (CGM) under standard conditions. Nanocomposites were introduced into the culture medium at final concentrations of 0.5, 1, 10, 100, and 1000 µg/mL, and their impact on cellular health was assessed following 72 h of incubation. The experiments were conducted in triplicate.

Cellular metabolic activity was evaluated using the Alamar Blue assay (TOX‐8; Sigma‐Aldrich/Merck Life Science Sp. z o.o., Poznan, Poland), following the manufacturer's instructions and previously described protocol [55]. Briefly, the culture medium was replaced with fresh CGM supplemented with 10% resazurin solution, and the cells were incubated for 2 h at 37°C in a CO_2_ incubator. Following incubation, 100 µL of the supernatant from each well was transferred to a 96‐well plate, and absorbance was measured using a multimode microplate reader (Spark, Tecan, Männedorf, Switzerland) at 600 nm (resazurin signal) and 690 nm (reference wavelength). The obtained values were expressed as ΔΔA, with blank samples included for baseline correction, and were used to determine the metabolic activity factor by comparing the metabolic activity of treated cells to that of the control cultures.

Following the screening of experimental cultures, hBMSCs were washed with HBSS and fixed with ice‐cold 4% paraformaldehyde (PFA; Sigma–Aldrich/Merck Life Science Sp. z o.o., Poznan, Poland) for 15 min at room temperature, followed by overnight incubation at 4°C to ensure complete fixation. After fixation, samples were thoroughly rinsed three times with HBSS containing 1% FBS to remove residual fixative. To enable intracellular staining, cell membranes were permeabilized by treating cultures with a 0.1% Triton X‐100 solution prepared in HBSS for 15 min at room temperature. Cytoskeletal actin filaments of cells were labeled using Atto‐488‐conjugated phalloidin (Sigma‐Aldrich/Merck Life Science Sp. z o.o., Poznan, Poland) diluted 1:800 in HBSS. The cells were incubated with the dye for 40 min at room temperature in the dark. For visualization of nuclei, hBMSCs were counterstained using 4′,6‐Diamidino‐2‐phenylindole dihydrochloride (DAPI) in mounting medium (ProLong Diamond Antifade Mountant with DAPI, Thermo Fisher Scientific, Warsaw, Poland). The morphology and ultrastructure of the cells were examined using epifluorescence microscopy (EpiFM; Axio Observer Z1/7, Zeiss, Oberkochen, Germany) at 100× magnification, with image acquisition performed using an Axiocam 503m S793 camera (Zeiss, Oberkochen, Germany). The analysis utilized three fluorescence channels for: (i) DAPI (excitation at 353 nm, emission at 465 nm; (ii) phalloidin (excitation at 493 nm, emission at 517 nm and incorporated biomaterials (excitation at 590 nm, emission at 618 nm).

Evaluation of Dy3+ ‐Doped Nanocomposites' Influence on Cellular Health–Comprehensive Investigation of Viability, Mitochondrial Activity, and Molecular Markers Influenced by Nanomaterials

4.10

For the functional assays, human bone marrow stromal cells (hBMSCs) were inoculated at a density of 2 × 10^4^ cells per well in 24‐well plates in 0.5 mL of complete growth medium (CGM) and treated with nanocomposites at a final concentration of 1 µg/mL for 72 h of incubation. Each experiment was performed in triplicate. After the assays, cells were harvested for cytometric‐based evaluation of cellular parameters and molecular biology tests.

Apoptosis was assessed by flow cytometry using the Dead Cell Apoptosis Kit with Annexin V for Flow Cytometry, while mitochondrial membrane potential was evaluated with the JC‐1 dye, both obtained from Thermo Fisher Scientific (Warsaw, Poland). Staining procedures were carried out according to the manufacturer's instructions. After staining, at least 10⁵ cells per sample were analyzed on a CytoFLEX flow cytometer (Beckman Coulter, CA, USA). Annexin V‐FITC and propidium iodide staining allowed differentiation between live, apoptotic, and necrotic cells, whereas JC‐1 fluorescence shifts were used to assess mitochondrial depolarization. Data acquisition and analysis were performed using CytExpert software, ensuring standardized gating strategies and compensation settings.

For RT‐qPCR, hBMSCs were homogenized using 1 mL of TRI Reagent (Sigma–Aldrich/Merck Life Science, Poznan, Poland) to determine transcript levels affected by the nanocomposites. Total RNA was extracted using the manufacturer's protocol, incorporating a modified phenol‐chloroform method by Chomczyński and Sacchi to ensure high yield and integrity [REF]. Concentration and purity were measured spectrophotometrically at 260/280 nm (DS‐11 Fx, Denovix, Wilmington, DE, USA). Before reverse transcription, 500 ng of total RNA was treated with DNase I (Thermo Fisher Scientific, Warsaw, Poland), eliminating any genomic DNA contamination. cDNA was synthesized using the Tetro cDNA Synthesis Kit (Bioline Reagents Limited, London, UK). For miRNA analysis, cDNA was synthesized using 375 ng of total RNA with the Mir‐X miRNA First‐Strand Synthesis Kit (Takara Bio Europe, Saint‐Germain‐en‐Laye, France). Genomic DNA removal and reverse transcription were conducted in a T100 Thermal Cycler (Bio‐Rad, Hercules, CA, USA), following standardized protocols to ensure reproducibility. The SensiFAST SYBR & Fluorescein Kit (Bioline Reagents Ltd., London, UK) was used to perform. RT‐qPCR with conditions optimized for specificity and efficiency. Each reaction (10 µL) contained 1 µL of cDNA and 0.4 µm primers (for mRNA detection) or 0.2 µm (for miRNA detection). Quantitative PCR was conducted on a CFX OPUS Real‐Time PCR Detection System (Bio‐Rad, Hercules, CA, USA), employing a thermal cycling profile to enhance amplification efficiency and minimize non‐specific products. Protocol details and reaction conditions have been previously described [55, 56]. Relative gene expression levels were determined using the RQ_MAX_ algorithm, with GAPDH and ACTB genes as the reference for mRNA normalization and U6 snRNA (Takara Bio) for miRNA normalization. Melting curve analysis was performed to confirm amplification specificity and exclude primer‐dimer formation. Primers used for RT‐qPCR are listed in Table S1.

Western blot analysis was performed using a previously established protocol to detect intracellularly accumulated proteins [56]. After the experiment, hBMSCs were lysed in ice‐cold RIPA buffer with 1% protease and phosphatase inhibitors, and then protein concentration was measured using the Pierce BCA Assay (all reagents derived from Thermo Fisher Scientific, Warsaw, Poland). Obtained samples were normalized (15 µg protein) and were incubated at 95°C for 5 min, mixed with 4× Laemmli buffer, and separated on 15% SDS‐PAGE at 100 V for 90 min. Proteins were transferred onto a PVDF membrane at 100 V for 60 min in 1× Transfer Buffer, and membranes were blocked (5% skim milk in TBS‐T). Reagents for SDS‐PAGE and blotting were derived from Bio‐Rad (Warsaw, Poland). The membranes were incubated overnight at 4°C with primary antibodies, followed by a 60 min secondary antibody incubation at room temperature, with five TBS‐T washes in between. Chemiluminescent signals were detected using the Bio‐Rad ChemiDoc XRS system with Pierce ECL substrate (Thermo Fisher Scientific, Warsaw, Poland) and analyzed via Image Lab Software (Bio‐Rad, Warsaw, Poland). Antibody details are in Table S2.

Statistical Analysis

4.11

Experimental data are presented as the mean ± standard deviation (SD), calculated from biological and technical replicates for each experiment. Statistical comparisons were performed using one‐way analysis of variance (ANOVA) followed by Dunnett's post hoc test, as appropriate. All statistical analyses were conducted using GraphPad Prism (version 8.20, CA, USA), with a significance threshold set at p < 0.05.

Photoluminescence Properties

4.12

Photoluminescence features were recorded by using an FLS980 Fluorescence Spectrometer from Edinburgh Instruments equipped with a 450 W Xenon lamp and a Hamamatsu R928P photomultiplier. The analysis includes emission spectra collected under the excitation wavelength 352 nm, excitation spectra recorded at 574 nm, and luminescence lifetime measured at room temperature associated with the transition ^4^F_9/2_→^6^H_13/2_ (*λ_ex_ * c = 351 nm; *λ_em_

• = 576 nm). Each spectrum was corrected during measurement in accordance with the characteristics of the intensity of the excitation source.

The Edinburgh Instruments FLS1000 Photoluminescence Spectrometer equipment with the Ultrafast photomultiplier MCP, range 200–850 nm detector, and InGaAs detector for decay time measurements, range 900–2550 nm, was used to record temperature‐dependent luminescence properties (excitation, emission, and luminescence kinetics). A Xenon lamp (450 W) and a Supercontinuum laser with a working range of 400–2000 nm were used as excitation sources. In order to set the ambient temperature, a Linkam heating‐cooling stage with a measurement range of 77–875 K was used.

Author Contributions

The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. S.T. designed the investigation; S.T. synthesized the samples; S.T., A.S., K.M., and D.S. designed the experiments; S.T., N.C., A.K., and D.S. performed the experiments; S.T. and N.C. did calculations; A.S. supervised in vitro studies and analyzed the data; K.M. performed the in vitro cultures and molecular biology experiments; J.S.‐Z. performed microscopic evaluations; S.T., A.S., K.M., and J.S.‐Z. prepared figures; N.C. prepared graphical abstract; A.S., K.M., and J.S.‐Z. analyzed data from in vitro studies; S.T., A.S., K.M., and J.S‐Z., D.S. wrote the material and method section; S.T. and A.S. drafted the manuscript; S.T.; A.S. and N.C. manuscript writing; R.J.W. supervision, conceptualization, resources, funding acquisition, and financial support, manuscript review & editing.

Funding

The authors would like to acknowledge the Foundation for Polish Science (FNP) no‐ START 083.2022 for supporting the research. Moreover, the authors would like to acknowledge the National Science Centre Poland (NCN) for financial support within the Projects ‘Biocompatible materials with theranostics’ properties for precision medical application’ (No. UMO‐2021/43/B/ST5/02960) under the National Science Centre Poland (NCN) and ‘Nanosized composites with biomimetics’ properties for precise medical application’ (BPN/BEK/2023/1/00309, Bekker programme) under the Polish National Agency for Academic Exchange, and Brazilian agency CNPq project no. 406079/2022‐6.

Conflicts of Interest

The authors declare no conflict of interest.

Supporting information

Supporting File: smll72143‐sup‐0001‐SuppMat.docx