Effect of calcination on physicochemical and biological properties of ion-modified nanohydroxyapatite for bone tissue engineering applications

doi:10.21203/rs.3.rs-3123280/v1

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Effect of calcination on physicochemical and biological properties of ion-modified nanohydroxyapatite for bone tissue engineering applications

https://doi.org/10.21203/rs.3.rs-3123280/v1

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published 16 Sep, 2023

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The study examines the effect of calcination at a temperature of 1200°C on the physicochemical and biological properties of nanohydroxyapatite (nanoHAP) substituted with magnesium (Mg²⁺), strontium (Sr²⁺), and zinc (Zn²⁺). The materials were characterized by Fourier-transform spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), and thermal analysis methods. Moreover, in vitro biological characterization, including cytocompatibility, cell proliferation, osteogenic potential, and reactive oxygen species production, was performed. The XRD results indicate that the ion substitution of nanoHAP has no effect on the apatite structure, and after calcination, β-tricalcium phosphate (β-TCP) is formed as an additional phase. SEM analysis showed that calcination induces the agglomeration of particles and changes in surface morphology. A decrease in the specific surface area and in the ion release rate was observed. Calcination and nanoHAP ion modification are beneficial for cell proliferation and osteoblast response and provide additional stimuli for cell commitment necessary for bone regeneration.

Physical sciences/Materials science/Biomaterials/Biomedical materials

Physical sciences/Materials science/Biomaterials/Implants

Physical sciences/Materials science/Biomaterials/Biomaterials cells

Physical sciences/Materials science/Nanoscale materials/Nanoparticles

Physical sciences/Materials science/Structural materials/Ceramics

nanohydroxyapatite

calcination

ion doping

osteoconduction

XRD

Hydroxyapatite (HAP) is a naturally occurring mineral that forms the mineral phase of bone. Synthetic HAP plays a significant role in bone regeneration. Literature data provide evidence that nanosized HAP promotes the formation of new bone tissue by increasing cell adhesion to their surface, enhancing cell differentiation towards the bone, and reducing inflammation [1]. Hydroxyapatite nanoparticles are also widely used as a component in the production of osteoconductive porous ceramic composites for orthopedic applications and bone regeneration. Improved mechanical properties due to the incorporation of crystalline nano- or micro-sized particles, characterize hydroxyapatite-reinforced composites. Depending on the production method, the addition of HAP may result in a different morphology and microstructure, significantly affecting the interaction between the living tissue and the implant, the exchange of nutrients through the pore structures, osteointegration, and osteoinduction [2, 3, 4]. The formation of chemical bonds between the implant and bone depends on partial enzymatic dissolution of the bioceramic surface, the ions released, and osteoclast activity. In effect, carbonated apatite precipitates at the implant-bone interface [5].

Among the several forms of apatites, there are biological apatites, found primarily in bones, teeth, and pathological tissues, e.g.,with calcifications, such as kidney stones or bladder stones. These organic apatites are composed not only of calcium and phosphorus but also contain trace elements such as Na⁺, Zn²⁺, Mg²⁺, K⁺, Si²⁺, and F^-, as well as carbonate and citrate ions [6, 7].

Another form of apatite is synthetic hydroxyapatite, composed of calcium and phosphorus in different stoichiometric molar ratios. Many studies demonstrate that the closer the Ca/P molar ratio is to the value of 1.67, the greater the stability of the material can be obtained inside the human body. On the other hand, better bioactivity can be achieved when the ratio < 1.67 [8, 9]. Hydroxyapatite can also be carbonated, and depending on the position of the carbonate group in the structure, various types of HAP can be distinguished, namely, HAP type A, with substituted hydroxide ions, HAP type B, with substituted phosphate ions, and a mixed AB type [10]. However, numerous scientific studies prove that HAP type B is essential for bone regeneration because it is similar to biological apatite [11].

The ability to fabricate HAP in different morphological and chemical forms draws attention to the proper choice of synthesis method and experimental conditions (temperature, time, and mixing speed) to acquire multifunctional HAP particles [1–3, 12]. Furthermore, some research concentrates on modifications that improve the physicochemical, mechanical, and biomedical properties of HAP [13, 14, 15].

Heating treatment conditions can enhance or weaken the properties of nanoparticles. Previous studies have indicated that higher temperatures (above 1000°C) favor the formation of other phases, such as β-TCP and tetracalcium phosphate [12]. As a result, calcination can not only aid in removing the excess water and other impurities from ceramics but also control phase formation [15]. Moreover, the ability for biological stimulation may be increased when additional modification of HAP is applied, and an example is ion substitution, where HAP can be doped with small amounts of ions found in natural bone minerals, e.g., strontium, zinc, or magnesium [16, 17].

Strontium has a two-way operation on bone and can work antagonistically. Low doses of strontium are involved in bone mineral metabolism by reducing the expression of genes related to osteogenesis, proliferation markers, and inhibiting apoptosis [18]. Sr²⁺ promotes osteogenesis, induces new bone formation, and relieves inflammation. But high Sr²⁺ concentrations have a toxic and inhibitory effect on osteoclast apoptosis [19]. Despite numerous studies on the topic, the mechanism of strontium’s impact on bone has not been fully recognized [20].

The crucial element, zinc, is an essential cofactor for enzymes involved in synthesizing DNA, RNA, and proteins. Zinc is located at the sites of tissue calcification, including osteons and calcified cartilage [21]. It takes part in the repair of cell membranes and the regeneration of the musculoskeletal matrix [22]. Zinc also stimulates bone formation and inhibits bone resorption through osteoclasts, which is consistent with the beneficial role of zinc in bone homeostasis and regeneration. Zinc's positive effects are related to osteoblasts' increased viability, osteogenic activity stimulation, bone healing, and antibacterial activity [23]. The optimal influence of zinc on osteoblasts occurs within a narrow dose range (1–50 µM), and doses above this range inhibit osteogenic activity [24, 25].

The last element mentioned, magnesium, is important for calcification and bone fragility [26, 27]. It is a critical component of our skeleton, and bones contain approximately 60% of the total body magnesium. In addition, magnesium embedded in hydroxyapatite influences the crystal size and strength [25]. Moreover, Mg²⁺ plays an essential role in the induction of the proliferation of osteoblasts and fibroblasts, thereby promoting osteogenesis and bone regeneration. Conversely, magnesium deficiency reduces bone stiffness, decreases osteoblast production, and promotes oxidative stress and subsequent bone loss, resulting in bone fragility [26, 27, 28].

By playing with the concentrations of functional elements or their types, different properties and structures of materials can be obtained, which are indirectly related to their biological potential. Additionally, calcination can enhance these properties by influencing structure, morphology, and phase composition.

This study aims to determine the effect of calcination at 1200°C and ion modification on nanoHAP physicochemical and biological properties, including biocompatibility, reactive oxygen species activity, and osteoconduction. For this purpose, we developed, fabricated, and characterized nanohydroxyapatite substituted with magnesium (Mg²⁺), strontium (Sr²⁺), and zinc (Zn²⁺). The novelty of this work lies in determining the properties of pure and ion-modified HAP as potential biomedical materials.

The method of producing modified HAPs proposed by us is easy, simple, and based on precipitation from aqueous solutions. On the other hand, our idea was to use further thermal processing of HAP to create a material with programmed physicochemical and biological properties.

The most crucial goal of this work is to show the synergistic effect of the two applied stages in HAP production: modification and heat treatment, which result in materials that may provide additional stimuli for cell commitment to serve bone regeneration. The results presented here are of great importance regarding the development of biomaterials. To the best of our knowledge, there are no literature reports on materials designed and obtained this way. These data provide valuable grounds for developing implantation biomaterials for bone lesion repair.

2.1 Synthesis and modification of nanohydroxyapatite

Non-substituted and substituted hydroxyapatites (Ca₁₀(PO₄)₆(OH)₂,) were prepared by the aqueous precipitation method from calcium hydroxide pure p.a. (Ca(OH)_2, Chempur) and 85% ortho-phosphoric acid, pure p.a. (H₃PO₄, Chempur) added dropwise over 45 min with constant stirring under alkaline conditions (pH = 11) by adding 25% ammonia solution (Chempur), if necessary. All reaction steps were performed at 50°C. The solution was stirred (450–800 rpm) for approximately 2–3 hours and then allowed to stand overnight at room temperature. The precipitate was rinsed 4–10 times with deionized water and centrifuged (3700 rpm for 2 min) until the excess of ammonia solution were removed. Purified nanoHAP was dried at 90°C for 12 h. Substituted hydroxyapatites were obtained by an analogous method, with the addition of precursors containing Mg²⁺ (Mg (NO₂)₃·6H₂O; Chempur, pure p.a.), Sr²⁺ ((Sr(NO₃)₂; Sigma-Aldrich pure p.a), and Zn²⁺ (Zn(NO₂)₃·6H₂O; Sigma-Aldrich, pure p.a.) to partially replace Ca²⁺ ions in the apatite structure. All compositions (Ca + X) /P molar ratio was 1.667, where X = Mg²⁺ or Sr²⁺ or Zn²⁺. The compositions of the synthesized samples and molar concentrations of the reactants used are given in Table 1.

Table 1

Molar concentrations of reactants and ions-calcium/phosphorus molar ratios.
Desired composition	Molar concentration of reactants					(Ca + X)/P molar ratio (X = Mg or Sr or Zn)
Desired composition	Ca (OH)₂	H₃PO₄	Mg (NO₃)₂ *6H₂0	Sr (NO₃)₂	Zn (NO₃)₂ *6H₂0	(Ca + X)/P molar ratio (X = Mg or Sr or Zn)
Ca ₁₀ (PO ₄) ₆ (OH) ₂	1	0.6	–	–	–	1.67
Ca ₉ Mg _0.1(PO ₄) ₆ (OH) ₂	0.9	0.6	0.1	–	–	1.67
Ca ₉ Sr _0.1 (PO ₄) ₆ (OH) ₂	0.9	0.6	–	0.1	–	1.67
Ca ₉ Zn _0.1 (PO ₄) ₆ (OH) ₂	0.99	0.6	–	–	0.1	1.67

The materials obtained were characterized, and the results were compared with pure HAP. A part of the nanoHAP powder was calcined at a temperature of 1200°C for 1 h (conditions: heating at 10°C/min to 1200°C, holding at 1200°C for 1 hour, cooling at 10°C/min to 20°C and then air-cooling to room temperature), to ensure that possible traces of ammonium nitrate were eliminated from the synthesized nanoHAP materials. The properties of materials after the calcination process were compared with non-calcinated nanoparticles.

2.2 Nanoparticles characterization

2.2.1 X-ray powder diffraction (XRD)

The phase composition of the synthesized powders was analyzed using the X-ray diffraction technique. The analyses were performed on a Bruker-AXS D8 DAVINCI diffractometer with a copper anode lamp in the Bragg–Brentano geometry. Diffractograms were recorded in the 2θ angular range from 5 to 120° (Cu Kα) at a measurement step of 0.01° and a measurement time 2 s/step. The crystalline phases were identified by comparing the registered diffractograms with the patterns in Crystallography Open Database (COD) using the DIFFRACplus EVA-SEARCH software. Quantitative analysis was performed using the Rietveld method in Topas v5.0 based on the published crystal structures (COD).

2.2.2 Scanning electron microscopy—energy dispersive spectroscopy (SEM/EDS)

Scanning electron microscopy with field emission was used to define the microstructure (shape and grain size) of the obtained hydroxyapatites (Nova NanoSEM 200, FEI). The samples were covered with a conductive material (20 nm gold film) using a sputter coater (EM SCD500, Leica). The hydroxyapatites imaging was performed in high vacuum conditions using a secondary electron detector at 10 kV accelerating voltage and at a magnification of 100,000x. Energy dispersive spectroscopy analysis was carried out to confirm the ion substitution on the uncoated samples (Octane Elect EDS, Edax, combined with SEM). EDS analysis was performed in low vacuum conditions at 15 kV accelerating voltage.

2.2.3 Specific surface area determination (BET)

The Brunauer–Emmett–Teller (BET) method was used to assess a specific surface area of the samples with Gemini VII 2390t Micromeritics analyzer. The analysis was based on the determination of 9 points nitrogen adsorption and desorption isotherms in the pressure range of 0.05–0.25 p/p°, where p and p° are the equilibrium and the saturation pressure of adsorbates at the temperature of adsorption, respectively. Before N₂ adsorption, the samples were degassed at 105°C in N₂ atmosphere for 1h to dry and purify the samples.

2.2.4 Fourier—transform infrared spectroscopy (FTIR)

The infrared spectra were recorded on Bruker TENSOR 27 instrument equipped with a DLaTGS detector. Powder samples were analyzed in the transmission mode with the following instrumental settings: wavenumber range of 400 − 4000 cm^− 1, number of scans 64, and spectral resolution 4 cm^− 1. Each sample was measured twice to check repeatability. The baseline correction procedure has been applied to the presented spectra with Opus 7.2 software.

2.2.5 Thermogravimetry Differential Thermal Analysis (TG—DTA)

The thermal properties of the powdered materials were analyzed with a STA F3 449 Jupiter® Netzsch thermal analyzer. TG–DTA experiments were performed under a dynamic flow of argon (70 mL/min) to investigate the particular mass loss due to the presence of a modifier. The 10 − 12 mg samples were heated from 30°C up to 1400°C at a heating rate of 10°C/min in an Al₂O₃ DTA pan with a lid and a hole. The measurements were repeated twice for each sample.

2.2.6 Ion Release Tests

The ion release was investigated by immersion of 1 g of each sample in 10 mL ultrapure water and further incubation. The incubation procedure was performed at 37°C in a separated closed flask for each sample, and after 1, 7, 21, and 42 days, the collected supernatant was filtered through 0.45 µm paper filter. Determination of selected elements was performed with inductively coupled plasma optical emission spectrometry (ICP—OES) using a Shimadzu ICPE 9800 spectrometer. The calibration solutions were prepared from Merck ICP multielement standard XVI storage solution (for Ca, Mg, Zn, Sr). The quantification limits of the method for the cations were 0.08 mg/L for Ca, 0.09 mg/L for Mg, 0.10 mg/L for Zn, and 0.02 mg/L for Sr. The conducted analytical tests meet the ISO 11885:2009 requirements. All experiments were made in triplicate.

3.1 Sterilization

Before the biological assays, all nanohydroxyapatite powders were sterilized by gamma radiation (35 kGy, ₆₀Co source) at the Institute of Applied Radiation Chemistry at the Lodz University of Technology (Lodz, Poland).

3.2 Cell culture conditions

The L929 (CCL-1) mouse skin fibroblasts recommended by the International Standard Organization (ISO) for the biological evaluation of biomaterials for medical applications were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Fibroblasts were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyCloneCytiva, Marlborough, MA, USA), penicillin (100 U/mL), and streptomycin (100 µg/mL) (Sigma-Aldrich, Darmstadt, Germany), in a humidified 5% CO₂ atmosphere at 37°C in cell culture incubator (Nuaire, Plymouth, MN, USA).

The human fetal osteoblastic cell line hFOB 1.19 (CRL-11372™) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F12 without phenol red (1:1 DMEM/F12 Modified; Gibco; Thermo Fisher Scientific, Waltham, MA, USA) containing a 0.3 mg/mL geneticin (Sigma-Aldrich, Saint Louis, MO, USA) and supplemented with a 10% fetal bovine serum. The cell cultures were incubated at 34°C in the incubator with a humidified air atmosphere containing 5% CO₂.

The confluent (80–90%) cell monolayers were periodically subcultured using a 0.5% trypsin-0.5 mM ethylenediaminetetraacetic acid tetrasodium salt (EDTA) solution (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The cell viability was established using a trypan blue exclusion assay. Only if the cell's viability exceeded 95%, the suspension was used for cell morphology and cell viability assessment.

3.3 In vitro cytocompatibility assay

To determine the influence of the prepared nanoHAP materials (at concentrations of 1 mg/mL, 5 mg/mL, and 10 mg/mL) on cell viability, in vitro cytosafety studies were carried out according to ISO-10993-5-2009 for testing components with potential application in biomedicine. Murine fibroblasts (L929, ATTC, Rockville, MD, USA) and human osteoblast cells (hFOB 1.19) followed by the MTT reduction assay were used as described previously [29]. Briefly, after the overnight incubation of the cells with the tested nanoHAP materials, 20 µL of MTT reagent (Sigma Aldrich) at a concentration of 5 mg/mL was introduced into each well and incubated for 4 hrs (5% CO₂, 37°C, > 90% humidity). In the next step, the plates were centrifuged (1200 rpm, 10 min.), and the supernatants were removed and replaced with 200 µL DMSO per well. After one minute of incubation at room temperature with shaking, the absorbance was measured at 570 nm using the Multiskan EX reader (Thermo Scientific).

3.4 Reactive oxygen species (ROS) production

For measuring changes in reactive oxygen species (ROS) levels in hFOB 1.19 cells after 1 and 24 hrs incubation with the bioceramics in concentrations of 1 mg/mL, 5 mg/mL, and 10 mg/mL, the H₂DCFDA fluorescent probe (Thermo Fisher Scientific) was used. H₂DCFDA, after deacetylation to H₂DCF (2',7'-dichlorodihydrofluorescein), is oxidized intracellularly to highly fluorescence-emitting 2',7'-dichlorofluorescein (DCF) [30]. Fluorescence was measured at the excitation wavelength of 495 nm and an emission wavelength of 525 nm using a SpectraMax® i3x Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA).

3.5 Osteoconductive potential

To assess the hFOB 1.19 osteoconductive potential, osteoblasts were grown in the milieu of a particular nanoHAP under osteoinductive conditions (39°C; differentiation medium: DMEM F-12 without phenol red, with 1% bovine serum, containing G148 geneticin and stimulants: β-glycerophosphate, ascorbic acid, dexamethasone, and the addition of the tested samples at a concentration of 5 mg/mL). As previously described, at the defined time points (7, 14, 21, and 28 days), the activity of alkaline phosphatase (ALP), cell proliferation, and the release of osteocalcin (OC) and interleukin-6 (IL-6) were quantified [31, 32]. Briefly, cell proliferation was evaluated based on DNA quantification by CyQuant® assay (Thermo Fisher Scientific). The activity of ALP was assessed by a p-NPP (para-nitrophenyl phosphate) hydrolysis assay. In contrast, the concentrations of OC and IL-6 in the culture media of hFOB 1.19 were determined by enzyme-linked immunosorbent assay (ELISA, R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The sensitivity of ELISA test for OC and IL-6 was 156.5 pg/mL and 9.38 pg/mL, respectively [32].

3.6 Statistical analysis

The statistical analysis was performed using GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA). Intergroup outcomes were compared for statistical significance using one-way or two-way ANOVA (analysis of variance). The differences were considered significant at the p-value < 0.05. The experiments were carried out in at least triplicate.

4.1. Phase composition determination

The X-ray diffraction patterns of the pure and substituted Sr²⁺, Mg²⁺, and Zn²⁺ nanohydroxyapatite are shown in Fig. 1a. All samples revealed a similar pattern characteristic for the hydroxyapatite phase (COD-9001233). The absence of additional phases may suggest that all ions have been successfully incorporated into the hydroxyapatite structure. The broad peaks of low intensity confirm the low crystallinity and small grain size of the prepared nanohydroxyapatites. After calcination, the hydroxyapatite peaks became sharper due to increased crystallinity and crystal growth. The calcination of HAP at 1200°C revealed differences in the thermal stability of the synthesized powders (Fig. 1a, Table 2). It was shown that substituting nanoHAP with Sr²⁺, Mg²⁺, or Zn²⁺ contributes to its faster decomposition, which is the indirect confirmation of nanoHAP ionic modification [17, 18]. The XRD patterns of thermally treated materials showed that the additional phase in the HAP structure is β-TCP. For nanoHAP, nanoHAP Mg 0.1, and nanoHAP Zn 0.1, diffraction peaks corresponding to CaO, MgO, and ZnO phases were identified, in addition to β-TCP. The highest proportion of β-TCP was observed in nanoHAP Mg 0.1 (44.88%).

Table 2

Phase composition of investigated powder before and after calcination at 1200°C.
non-calcinated			calcinated (1200°C)
Material	Phase (COD)	Content [%]	Material	Phase (COD)	Content [%]
nanoHAP control	Hydroxylapatite (9001233) Ca₁₀(OH)₂(PO₄)₆	100	nanoHAP 1200°C control	Hydroxylapatite (9001233) Ca₁₀(OH)₂(PO₄)₆	93.77
				Tricalcium phosphate (1517238) Ca₃(PO₄)₂	5.69
				CaO (9006694)	0.54
nanoHAP Sr 0.1		100	nanoHAP Sr 0.1 1200°C	Hydroxylapatite (9001233) Ca₁₀(OH)₂(PO₄)₆	88.64
nanoHAP Sr 0.1		100	nanoHAP Sr 0.1 1200°C	Tricalcium phosphate (1517238) Ca₃(PO₄)₂	11.36
nanoHAP Mg 0.1		100	nanoHAP Mg 0.1 1200°C	Hydroxylapatite (9001233) Ca₁₀(OH)₂(PO₄)₆	50.97
				Whitlockite (9012415) Ca₉MgH(PO₄)₇	44.88
				MgO (4001314)	4.16
nanoHAP Zn 0.1		100	nanoHAP Zn 0.1 1200°C	Hydroxylapatite (9001233) Ca₁₀(OH)₂(PO₄)₆	73.76
				Tricalcium phosphate (1517238) Ca₃(PO₄)₂	25.61
				ZnO (2107059)	0.63

4.1 Morphological Characterization

The scanning electron microscope (SEM) indicated a globular morphology and agglomeration behavior, typical of precipitated nanoHAPs, and confirmed the nominal substitution of Mg²⁺ or Sr²⁺ or Zn²⁺ for both non-calcinated and calcinated nanoparticles. The chemical composition of the synthesized products was determined by the energy dispersive spectroscopy (EDS) analysis shown in Fig. 2. The size of non-calcinated sphere-like shape particles ranged 36.5–49.6 nm for pure nanoHAP, 40.8 nm – 48.1 nm for Mg ²⁺, 53.6–61.3 nm for Sr ²⁺, and 40.9–49.5 nm for Zn²⁺doped nanoHAP, which confirms the nanometric size of the hydroxyapatite particles. Calcinated particles had a size of 108.0 nm – 1.08 µm for nanoHAP; 114.9 nm – 984.4 nm for Mg²⁺, 46.7 nm – 937.3 nm for Sr ²⁺, and 39.1 nm – 971.3 nm for Zn²⁺ doped nanoHAP. The SEM micrographs (Fig. 1b) confirm that calcination affected the morphology of hydroxyapatites and resulted in the diffusion of the particles to form a bigger irregular structure with a nearly round shape and interconnected fine particles and pores.

4.2 Specific surface area (BET)

The BET theory explains the physical adsorption-desorption of gas molecules on a solid surface and represents the basis for determining the specific surface area [19]. BET analysis results for the specific surface area are presented in Table 3 for materials before and after the calcination. The results of the BET analysis show that the surface area of the nanoHAP substituted with Zn²⁺ was greater (100.1 m²/g) than nanoHAP substituted with Mg²⁺ (76.4 m²/g) and nanoHAP substituted with Sr²⁺ (66.1 m²/g). However, after the calcination process, the surface area dropped drastically in the obtained samples, and the highest value was determined for the material with magnesium presence.

Table 3

Specific surface areas of materials.
Material	Specific surface area [m²/g]
Material	Non-calcinated	Calcinated at 1200°C
nanoHAP	80.7	0.3310
nanoHAP Sr 0.1	66.1	0.3705
nanoHAP Zn 0.1	100.1	0.6005
nanoHAP Mg 0.1	76.4	1.3333

Table 4

IR bands assignments for nanohydroxyapatites before and after calcination (vw-very weak, w-weak, sh-shoulder, nd-not determined).
Band assignment	HAP	HAP 1200°C	HAP Sr 0.1	Hap Sr 0.1 1200°C	HAP Zn 0.1	HAP Zn 0.1 1200°C	HAP Mg 0.1	HAP Mg 0.1 1200°C
OH surface	-	3734	-	3733	-	3732	-	3733
OH surface	3568	3571	3565	3571	3568	3572	3572	3672
water	3422	3434	3428	3432	3425	3409	3412	3414
water	3237 w	nd	3237 w	3247	3248 w	nd	3233	nd
CH₃,CH₂	2977/2940/2904	2974/2942/2886	2977/2944/2904	2975/2945/2880	2974/2945/2901	2972/2945/2882	2974/2946/2897	2972/2945/2882
overtonesCO₃²⁻ and ν₃/ν₁ PO₄	1998	2003	1992	1995	2000	2002	2004	2004
HPO₄²⁻	-	1765/1703 vw	-	1760/1706 vw	-	1760/1716 vw	-	1761/1705vw
Water O-H	-	1615	-	1618	-	-	-	-
Water O-H	1634	-	1634	-	1637	1623	1637	1623
C-O str. P-Ar	1496 w	-	-	~ 1485	1494	nd	~ 1488 vw	-
ν3 C-O (CO₃²⁻) CH2 bending	1453	-	1456	-	~ 1460 vw	nd	-	-
ν3 C-O (CO₃²⁻) CH2 bending	1421	-	1421	-	1421	nd	1422	-
NO³⁻ HPO₄²⁻	-	1382	1385	-	1385	nd	1385	~ 1372
P-O	-	-	-	-	-	-	-	1117
ν3 asym. PO₄³⁻	1092	1091	1094 sh	1090	1094 sh	1090	1101 sh	1090
ν3 asym. PO₄³⁻ ideal crystal	1033	1048	1031	1047	1035	1045	1034	1044
asym. str. P-O	962	961	960	958	963	970 w	961	962 (973/948)
CO₃²⁻	874	-	875	-	875	-	874
NO₃⁻	-	-	839	-	838	-	838	839 vw
sym.str. P-O-C	-	-	-	-	-	814	-	-
str. OH⁻ labile PO₄³⁻	sh636	632	-	630 w	~ 634 sh	-	634 sh	631
603 + 562 + 473 ν₄ str. PO₄³⁻	603	601	602	600	603	602	603	602
603 + 562 + 473 ν₄ str. PO₄³⁻	565	570	564	569	565	571	565	572
HPO₄²⁻, P₂O₇⁴⁻	505	-	-	-	-	498	-	508
ν₂ PO₄³⁻	468	-	469	467	469	-	468	-
H₂PO⁴⁻, or P₂O₇⁴⁻	-	~ 438	-	427	-	-	-	-

Table 5

Selected IR bands of apatites after calcination together with their assignments and position.
Sample	Wavenumber (cm^− 1) and the assignment
Sample	~ 3420 OH	1091 PO₄³⁻	1048 PO₄³⁻	961 P-O	633 OH-	603 + 562 + 473 PO₄³⁻	505/498 PO/PO
NanoHAP 1200°C	3434	1091	1048	961	632	601/570	476
NanoHAP Mg 0.1 1200°C	3414	1090	1044	962	631	602/572	508
NanoHAP Sr 0.1 1200°C	3432	1090	1047	958	630	600/569	467
NanoHAP Zn 0.1 1200°C	3409	1090	1045	-	-	602/571	505/498

Table 6

Thermal parameters for the materials before calcination (Δm – mass loss, T_dtg – extremum for DTG peaks, T_dta – extremum for DTA peaks). nd-not determined, 01-crucible without lid, 02-crucible with lid.
Sample	Δm (wt.%)	Solid reisude (wt.%)	T_dtg (°C)	T_dta (°C)
nanoHAP-01	-4,28/-0,71	95.01	118	685/715/1368
nanoHAP-02	-4,24/-1,56	94.24	116	699/716/1375
nanoHAP Mg 0.1	-6,86/-2,49/-1,76	88.89	116	nd
nanoHAP Sr 0.1	-5,73/-4,20	90.07	117	nd
nanoHAP Zn 0.1	-5,67/-3,65	9.,68	103	nd

4.3 Fourier—transform infrared spectroscopy (FTIR).

Figure 2a shows the FTIR spectra of nanohydroxyapatite before calcination. Almost all characteristic bands for hydroxyapatite can be identified [33, 34]. The medium intense band at about 3572 cm^− 1 is due to the vibrations of hydroxyl groups on the HAP surface. The bands observed in the range of 3237–3422 cm^− 1 and at 1634 cm^− 1 are related to the presence of water. Medium bands at about 1453 cm^− 1, 1421 cm^− 1, and 874 cm^− 1 are assigned to ν₃ C-O (CO₃²⁻) vibrations. The four vibrational modes associated with phosphate ions were identified at ν₁ 962 cm^− 1, ν₂ 468 cm^− 1, ν₃ 1092 + 1033 cm^− 1, ν₄ 603 + 562 cm^− 1, while the band at 636 cm^− 1can be assigned both to the O—H and labile PO₄³⁻ [35]. The absence of band at about 1190 cm^− 1 may result from different phosphate chemical environments.

The Mg, Sr, and Zn nitrate precursors partially replaced calcium ions in the apatite structure. In the spectra of modified hydroxyapatite before calcination, additional peaks were observed at 1385 cm^− 1 and 839 cm^− 1, which is related to the presence of unreacted nitrates (Fig. 2a-b). Therefore, the signals of phosphates are partially masked and can be distinguished only after thermal treatment and nitrate degradation (temperature above 500°C). In nanoHAPs doped with magnesium, a weak band at 838 cm^− 1 may be related to MgO presence. Other characteristic bands for MgO were not observed or overlapped with similar bands, i.e., the signals at 1385 cm^− 1 and 3412 cm^− 1.

To follow the structural changes during calcination, FTIR spectra of the material after thermal treatment were compared and are presented in Fig. 2c. After calcination at 1200°C some specific bands disappear, for example, the most prominent ones for nitrates, 1385/839 cm^− 1, and carbonates, 1453/1421 cm^− 1 and 875 cm^− 1. This is consistent, as at a temperature above 500°C thermal degradation of stabile ions occurs. Moreover, some bands shift by ± 4 cm-1; for example, the band at 1033 cm-1 and the weak band around 1382 cm-1 are shifted. These effects are probably related to the differences between nanoHAP materials regarding hydroxyl and phosphate bands that may suggest a different substitution of HAP sites as well as the formation of different apatites. Calcination leads to the condensation of surface hydroxyl groups with the formation of water, which was proved by the presence of bands at 3734 cm^− 1 followed by the ones above 3400 cm^− 1.

4.4 Thermal behavior

The thermal behavior of nanoHAP was investigated during TG–DTA experiments in the inert atmosphere of argon. Thermogravimetric (TG) curve of the unmodified nanoHAP showed an initial mass loss of about 4 wt.% related to the evaporation of physically adsorbed water on the surface of the particles and chemically adsorbed water, as well as crystal water and lattice water lost till 400°C. It is impossible to distinguish mass losses in this region, most probably because the measurements were conducted with lids on the top of the Al₂O₃ crucible. Despite the lid has a small hole to ensure the gas flow, some limitations related to the gas diffusion into the sample are possible. Simultaneously, the use of lids prevents the nanoparticles’ escape from the crucible. TG curves for the modified ceramics show higher mass loss than those for nanoHAP, indicating the decomposition of residual reagents (nitrates) and possibly the constituents of the nanoHAP (carbonates, hydroxyls). For HAP, we observed that dehydroxylation of surface phosphorous occurs in the temperature range of 450–730°C, whereas the dehydroxylation of hydroxyapatite can take place a thigher temperature of 730–1020°C [36]. It has been reported that dehydroxylation of HAP starts at about 800°C and leads to the decomposition of HAP to oxyapatite Ca₁₀(PO₄)₆O (Fig. 3) [37, 38].

4.5 Analysis of the Ion Release Profile

Figure 4 shows the release profile of Ca²⁺ for nanoHAP and nanoHAP Zn 0.1 and the profile of Mg²⁺ and Sr²⁺ ions released from nanoHAP Sr 0.1, nanoHAP Mg 0.1, both in non-calcinated and calcinated samples. The test was performed in static conditions (without changing the medium) in deionized water at 37°C for 1, 7, 21, and 42 days. Unfortunately, zinc ion release levels were under the detection limit of ICP-OES and could not be monitored. That is why for zinc-modified HAP, only calcium release was determined.

The results demonstrated that if the nanoHAP was doped with zinc ions, the level of calcium release increased almost six times (Fig. 4a-b). For unmodified HAP, an interesting fact of a significant increase of calcium release after 42 days of incubation was observed, which needs to be further clarified. The calcination process led to a meaningful decrease in the calcium release level, probably due to the stabilization of ions in the hydroxyapatite structure and reduction of the surface area of the tested powders.

Non-calcinated nanoHAP Mg 0.1 showed a burst release of Mg²⁺ ions from the 1st until the 7th day of incubation, ranging from 4000 ppm to 8000 ppm. After 7 days, the release of Mg²⁺ was decreased to 4200 ppm. Long-term magnesium release was maintained at a similar level until the end of the experiment (Fig. 4c). The calcination in this case, also leads to a significant decrease in magnesium release. However, after 21 days of incubation, an increase was observed again. Regarding controlled ion supplementation, this is a very promising phenomenon.

For HAP modified with Sr²⁺(Fig. 4d), significantly lower values of ion release were observed, and this level increases slightly until 21 days of incubation, while after this time decreases to the starting level Analysis of the calcinated HAP substituted with Sr showed that ion release raised to 42 days of incubation, and the highest value jump was registered after 21 days of incubation.

5.1 In vitro cytocompatibility

The effect of non-calcinated and calcinated (1200°C) nanoHAP materials on the metabolic activity of L929 fibroblasts (Fig. 5a) and human hFOB 1.19 osteoblasts (Fig. 5b), was investigated.

The prepared materials were tested in solutions of 1 mg/mL, 5 mg/mL, and 10 mg/mL. The viability of mouse fibroblasts and human osteoblasts incubated for 24 hrs in the presence of non-calcinated materials was dose-dependent. Calcination of the samples at 1200°C caused a notable increase in the viability of cell (L929 and hFOB) cells, and there was no dose dependence, except for nanoHAP Zn 0.1. The non-calcinated samples of nanoHAP (1 and 5 mg/mL), nanoHAP Zn 0.1 (1 mg/mL), nanoHAP Sr 0.1 (1, 5 and 10 mg/mL), nanoHAP Mg 0.1 (1, 5 and 10 mg/mL) and calcinated samples of nanoHAP 1200℃ (1, 5 and 10 mg/mL), nanoHAP Zn 0.1 1200℃ (1, 5 and 5 mg/mL), nanoHAP Sr 0.1 1200℃ (1, 5 and 10 mg/mL), nanoHAP Mg 0.1 1200℃ (1, 5 and 10 mg/mL) did not cause a decrease in L929 cells’ metabolic activity below 70%. These materials were thus confirmed to be cytocompatible on an in vitro levels.

5.2 ROS production

To assess nanoHAP's influence on human osteoblasts regarding reactive oxygen species (ROS) production, H₂DCFDA probe was used. The results presented in Fig. 6a-b show that 1 h incubation of hFOB 1.19 cells in the presence of nanoHAP Mg 0.1, both raw and calcinated at 1200°C, significantly increased ROS levels in a dose-dependent manner. However, after 24 hrs, the increase in the ROS level was the highest only after incubation with non-calcinated nanoHAP Mg 0.1 in the concentration of 10 mg/mL. A significant increase of over 150% in ROS production has also been observed for calcinated nanoHAPs modified with strontium (1 and 5 mg/mL) after 1 h of incubation. The observed effect was not dose-dependent. For nanoHAP 1 mg/mL and nanoHAP Zn 0.1 10 mg/mL, a statistically significant decrease in the ROS levels after 24 hrs of incubation was noticed.

5.3 Osteoblast differentiation and proliferation

The osteoconductive potential of nanohydroxyapatites was analyzed and presented in Fig. 7. The results indicate that the calcination of the samples at 1200°C led to a significant increase in ALP secretion by hFOB 1.19 compared to the concentration of this osteoblastic marker detected in cell cultures incubated with the non-calcinated samples (Fig. 7a, g, j). The amounts of ALP detected in 28-day osteoblast cultures incubated with calcinated nanoHAP and ion-modified nanoHAP reached: 1.7 ± 0.02 IU/mL (nanoHAP), 2.4 ± 0.5 IU/mL (nanoHAP Sr 0.1), 1.9 ± 0.3 IU/mL (nanoHAP Mg 0.1), and were significantly higher (p < 0.05) as compared to ALP level detected in the presence of non-calcinated specimens: 0.8 ± 0.02 IU/mL (nanoHAP), 0.8 ± 0.05 IU/mL (nanoHAP Sr 0.1), 1.0 ± 0.01 IU/mL (nanoHAP Mg 0.1). Interestingly, no differences between the levels of ALP produced by osteoblasts cultured with non-calcinated neither calcinated nanoHAPs modified with Zn (Fig. 7d) were observed. Moreover, ALP production was lower than the activity detected in the control cell cultures at all time points. The production of osteocalcin, another key marker of osteoblast differentiation, was significantly higher in hFOB 1.19 cell cultures incubated with calcinated nanoHAPs at all time points, compared to the level of OC detected in cell cultures exposed to non-calcinated samples, 599.3 ± 21.6 pg/mL and 157.8 ± 8.9 pg/mL, p < 0.0001 (on day 7) and 783.3 ± 0.9 pg/mL and 584.0 ± 9.3 pg/mL, p < 0.0001 (on day 28), respectively (Fig. 7b). Significant differences were also noticed between the calcined and non-calcined nanoHAPs with Zn, Sr, and Mg, which confirmed the beneficial effect of calcination at 1200°C and ionic modification on increased OC production in hFOB 1.19 cells (Fig. 7e, f, k).

Interestingly, the IL-6 production decreased throughout the entire course of the experiment. However, at all time points, the level of IL-6 produced by hFOB 1.19 cultured on calcinated nanoHAPs was higher (63.0 ± 5.3 pg/mL) than that detected in cultures exposed to non-calcinated samples (11.6 ± 0.2 pg/mL), p = 0.03 (Fig. 7c). This trend in IL-6 secretion was also demonstrated for calcinated vs. non-calcinated nanoHAPs modified with Zn, Sr, and Mg (Fig. 7c, f, i, l).

We have quantified DNA after different time points to assess the influence of fabricated nanoHAP materials and their modification on the long-term viability of osteoblast cultures. In this assay, the fluorescence intensity of DNA corresponds to the number of cells and thus the measurement of cell proliferation is possible. The results indicated that calcination of the samples at 1200°C promoted efficient osteoblast proliferation (Fig. 8). The mean number of cells (MNoC) present within calcinated nanoHAPs after 28 days were significantly higher (p < 0.05) than in non-calcinated specimens: nanoHAP (5.9×10²±1.2×10² vs. 2.6×10⁴±7.2×10), nanoHAP Zn 0.1 (4.3×10²±8.3×10² vs. 1.9×10⁴±1.1×10³), nanoHAP Sr 0.1 (5.0×10²±1.1×10² vs. 1.8×10⁴±3.2×10³), and nanoHAP Mg 0.1 (4.4×10²±1.3×10² vs. 1.7×10⁴±2.1×10³).

Currently, nanohydroxyapatite is widely used in bone regeneration, therefore, it is crucial to investigate its possible further improvements. Thus, we aimed to understand the influence of ion doping and the calcination process on HAP properties and biological activity. In this paper, we present the results of the characterization of nanohydroxyapatite synthesized by precipitation from an aqueous solution, and further modified with Mg²⁺, Sr²⁺, and Zn²⁺. Moreover, the effect of calcination at 1200°C was correlated with the influence of the ion modification.

We initiated our study with a thorough characterization of the chemical structure, phase composition, and microstructure of the nanoHAP and its modifications. The FTIR data confirmed the presence of characteristic bands for the designed hydroxyapatite. The XRD results indicated that incorporating Sr²⁺, Mg²⁺, and Zn²⁺ into nanoHAP stabilized the hydroxyapatite structure. These results agree with other data, which showed a preferential substitution of cations in the Ca(II) position [39]. We observed that the calcination process led to significant structural changes, as proved by FTIR (disappearance of particular bands) and XRD (formation of additional phases). After calcination, the quantity of HAP decreases, and β-TCP is formed as the second most prominent phase. The highest proportion of β-TCP was observed in nanoHAP Mg 0.1 (44.88%), then in nanoHAP Zn 0.1 (25.61%), nanoHAP Sr 0.1 (11.36%), and finally the smallest in pure nanoHAP (5.69%). In calcinated nanoHAP, nanoHAP Mg 0.1, and nanoHAP Zn 0.1, diffraction peaks corresponding to CaO, MgO, and ZnO phases were identified, in addition to β-TCP. This is in line with previous reports, which showed that at higher temperatures (above 1000°C), hydroxyapatite could convert to TCP and calcium oxide (CaO) phases following the equation below [39, 40]:

Ca₁₀(PO₄)₆(OH)₂→ 3Ca₃(PO₄)₂ + CaO + H₂O

The coexistence of HAP with β-TCP and CaO strongly depends on the change in Ca/P molar ratio relative to the stoichiometric value of 1.667; even a slight variation in Ca/P ratio may result in significantly different phase composition [41]. A high proportion of β-TCP in the structure, related to the different 1.667 Ca/P ratio, can worsen the biological properties of bioceramics. On the other hand, the presence of CaO and a higher Ca/P ratio improve biological activity [40, 42]. Moreover, it was shown that ionic substitution destabilizes the hydroxyapatite structure and favors its thermal conversion into β-tricalcium phosphate even at a lower temperature [43–49]. Furthermore, previous reports showed that as the calcination temperature increases, more of the β-TCP phase is formed, which is in line with our results [50].

No noteworthy observation comes from the thermal analysis of the materials, which may indicate that undetectable amounts of modifiers are present in the structure of nanopowders. The nano-sized material is tough to analyze in TG-DTA experiments, where the dynamic gas flow can cause the nanoparticles' entrainment by the carrier gas. In addition, the bulk material inside the crucible can create an air cushion and interfere with the data recording. The only visible effect is that some hydroxyl groups escape from the structure after calcination (mostly loosely bound water molecules).

Another essential material characteristic is its morphology, and this was investigated using SEM equipped with an EDS detector to check the effect of modification on the chemical composition of HAPs. The SEM micrographs demonstrate that calcination and ion substitution affect the morphology of hydroxyapatite. The calcination resulted in the diffusion of the particles to form larger, more irregular structures with a nearly round shape and interconnected fine particles and pores. Similar results were presented by Aina et al. [16, 51], Mocanu et al. [17], and Ofudje et al. [52], who observed the growth of various round-shape crystals after the calcination and substitution of magnesium, strontium, or zinc ions in the structure of apatite. These various-size crystals with irregular fine-like morphology of nanoHAP particles open up new perspectives forpromoting tissue growth during bone implant application [13].

The results of the ion release test for the nanopowders showed that both ion modification and thermal treatment strongly affected the nanopowders' behavior. Following thermal treatment at 1200°C, the raw nanoHAPs changed their virgin performance, and the ion release dropped significantly in all variants. This might have depended on several factors. Above all, a significant reduction in the specific surface area of nanoHAP grains after calcination may have mattered. The calcination-induced decrease in the size of the material's specific surface area capable of ion exchange seemed critical in reducing the ions’ release.

The most prominent and only significant release of ions was observed for magnesium-modified HAP, but not for non-calcinated materials. Calcination led to a gradually increasing Mg²⁺ release over time. This result, apart from the size of the specific surface area, may be related to the phase composition of the material received after calcination. Calcinated Mg-modified nanoHAP contains two phases of calcium phosphate: HAP and β-TCP, with the largest proportion of β-TCP among all obtained nanoHAPs. According to the literature, β-TCP is a well-resorbable material [53–54], and HAP is regarded as a non-resorbable material [56]. Thus, we suppose that the rate of ion release could be affected by the degradation/resorption capacity of the obtained material, and the more β-TCP, the more ions are released. In addition, the XRD data detected the magnesium oxide form, which is the most easily and fast released from materials. Non-calcinated material showed an initial burst release of Mg²⁺ ions, followed by a noticeable decline. These results are consistent with the data presented by Khoshzaban et al. [57] and Sprio [58], and this phenomenon could be related to the release of loosely bonded ions, resulting in faster ion loss rates at the initial stages of incubation [17, 18].While analyzing ion release profiles from strontium- and magnesium-modified nanoHAPs, we noticed that the release rates of Sr²⁺ were significantly slower than those of Mg²⁺. This may be due to much stronger Sr²⁺than Mg²⁺bonding to the nanoHAP structure [59]. The material most distinct from the others was the one with zinc ions in the structure. The amount of zinc ions released was under the instrument detection limit, and only calcium ions’ release could be followed. A similar situation was observed by Sprio et al. [59] and Mocanu A. [17], even if the experiment was conducted for 90 days. In the case of the zinc-modified HAP, we observed calcium release. Calcium release from the calcinated Zn-modified nanoHAP was relatively stable, while surprisingly, in the non-calcinated Zn-modified HAP the calcium release level was significant higher. Unfortunately, zinc ion release levels which were under detection. The most plausible explanation for these results, as suggested by Li [60], is that the presence of PO43- groups in a solution can affect the dissolution of Zn²⁺ by inhibiting their release and by absorbing them on the surface of ZnO particles. Considering the behavior of Zn-modified HAP, we may also speculate that Zn²⁺ ions are located in energetically stable substitutional positions of the HAP and β-TCP phases [60, 62].

To check the effect of HAP modifications, we also performed analyses of the specific surface area and porosity of materials. The results showed that the highest surface area among all tested materials characterized Zn-modified material. Studies in this area are lacking, and further research is needed to fully describe the effect of zinc modification on the physicochemical and biological properties of biomaterials.

The main objective of our study was to assess the outcome of doping ions on the biological characteristics and bioactivity of nanoHAP. Our results showed that nanoHAP modified with Mg, Sr, and Zn, after calcination promotes the growth and proliferation of cells better than non-calcinated materials. Moreover, the advantageous effect of ion-modified nanoHAP on osteoblast response is associated with improved biocompatibility and cell activity, which infers that metabolism is affected by nanoHAP doping ions during bone remodeling [64]. Both early and late osteogenesis markers (osteocalcin and ALP, respectively) showed that calcination promoted high cell activity in osteoblast cell culture. This result is of particular importance in the context of osteoconduction and for a better understanding of the role of ion substitution in HAP properties. Similarly, Mocanu et al. [17], Rapuntean S et al. [64]. de Lima et al. [65], and Gnaneshwar et al. [66] reported that Sr-, Mg-, or Zn-substituted hydroxyapatite could improve osteoconduction and enhance osteoblast proliferation.

Additionally, we examined the proinflammatory cytokine IL-6, which may play opposing roles. In the early stage, during fracture healing and support regeneration, it contributes to bone remodeling, but its excessive production may lead to chronic pathological inflammation [36, 67]. Moreover, IL-6 may influence osteogenic differentiation by inhibiting the secretion of osteogenic markers or promoting osteoblast proliferation [68]. Our analyses demonstrated that calcinated materials cause a gradual decrease in IL-6 secretion. However, with non-calcinated materials, especially zinc-modified HAP, lower levels of IL-6 were observed. A similar trend for IL-6 concentration was registered for raw nanoHAPs and magnesium- and strontium-modified nanoHAPs. These results are similar to other available data and show that the secretion of IL-6 does not affect the production of osteocalcin and alkaline phosphatase by osteoblastic cells [17, 68–71].

Additionally, we analyzed the ROS production in the presence of the obtained materials, as ROS values increase considerably during tissue regeneration, triggering a local inflammatory response that is essential for accelerating wound healing and restoring homeostasis [72]. ROS may be used as a parameter for controlling biological properties and enhancing cell activity. The materials reacting in an oxidative microenvironment are promising therapeutic substances that may be developed as smart drug delivery systems, contributing to the acceleration of bone regeneration [73]. Our analysis shows that calcinated magnesium- and strontium-modified HAP has such potential.

To summarize, our data contribute to developing advanced biomaterials for application in bone regeneration and replacement. We show that substitution elements, such as Mg²⁺, Sr²⁺, or Zn²⁺, make the nanoHAP a multifunctional material ready for further applications or investigations as a component of more futuristic biomaterials. I addition calcination may additionally ameliorate the final properties of bioceramics.

This paper presents a detailed study on the fabrication and characterization of nanohydroxyapatite for biomedical applications. NanoHAP ion modification and calcination at 1200°C proved to be beneficial for the structural and biological characteristics of the biomaterial. Both processes influence the crystallinity, specific surface area, and morphology of the nanopowders. Moreover, calcination induces the formation of new phases in the bioceramic structure that significantly affect the ion release level, thus influencing biological activity. All designed materials except those with Zn²⁺ remained cytocompatible. Importantly, by calcinating of ion-modified HAPs, valuable biological properties were achieved, such as stimulation of osteoblast proliferation and induction of their differentiation. Combining calcination and ionic modification of nanoHAP yields exciting results and opens up new possibilities for creating biomaterials with programmed properties to make them suitable for biological and medical applications.

In conclusion, modifying hydroxyapatites with specific divalent cations is promising for the development of advanced multifunctional materials for bone regeneration and replacement.

HAP – hydroxyapatite; nanoHAP – nanohydroxyapatite, β-TCP – β-tricalcium phosphate

Contribution

Agata Kurzyk: Study concept, Collection of data, Elaboration of data (synthesis and modification of hydroxyapatite, release ions), Data analysis, Graphical abstract, Writing and Editing the Manuscript, Aleksandra Szwed-Georgiou: Collection of data, Elaboration of data (in vitro cell analysis), Writing and Editing Original Draft Preparation, Joanna Pagacz: Collection of data, Elaboration of data (FTIR, TG-DTA), Writing and Editing Original Draft Preparation, Agnieszka Antosik: Collection of data, Elaboration of data (X-ray diffraction, BET), Paulina Tymowicz-Grzyb: Investigation, Collection of data (SEM observations), Anna Gerle: Investigation, Collection of data (ICP release ions), Piotr Szterner: Methodology (synthesis and modification of hydroxyapatite), Collection of data, Marcin Włodarczyk: Collection of data, Elaboration of data (in vitro cell analysis), Przemysław Płociński.: Conceptualization of Biological Analyses, Writing and Editing Original Draft Preparation, Mateusz M. Urbaniak: Investigation, Collection of data (ROS), Karolina Rudnicka: Conceptualization of Biological Analyses, Writing and Editing Original Draft Preparation, Funding Acquisition, Monika Biernat: Conceptualization, Supervision, Writing - original draft, Funding Acquisition. All authors have read and agreed to the published version of the manuscript.

Declaration of Competing Interests

None.

Acknowledgments

This work was supported by the TEAM - NET programme of the Foundation for Polish Science financed by the European Union under the European Regional Development Fund („The Multifunctional composites biologically active for applications in regenerative medicine of bone system” project [POIR.04.04.00-00-16D7/18].

Data Availability Statement

The data generated during this study are available at ŁUKASIEWICZ Research Network Institute of Ceramics and Building Materials, Center of Ceramic and Concrete in Warsaw, Biomaterials Research Group, Postępu 9, Warsaw, 02-676, Poland. Biological research data are available at University of Lodz, Faculty of Biology and Environmental Protection, Department of Immunology and Infectious Biology, Banacha 12/16, 90-237 Lodz, Poland. All data are available from the corresponding author upon request.

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Effect of calcination on physicochemical and biological properties of ion-modified nanohydroxyapatite for bone tissue engineering applications

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Journal Publication

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Synthesis and modification of nanohydroxyapatite

2.2 Nanoparticles characterization

2.2.1 X-ray powder diffraction (XRD)

2.2.2 Scanning electron microscopy—energy dispersive spectroscopy (SEM/EDS)

2.2.3 Specific surface area determination (BET)

2.2.4 Fourier—transform infrared spectroscopy (FTIR)

2.2.5 Thermogravimetry Differential Thermal Analysis (TG—DTA)

2.2.6 Ion Release Tests

3. In vitro biological characterization

3.1 Sterilization

3.2 Cell culture conditions

3.3 In vitro cytocompatibility assay

3.4 Reactive oxygen species (ROS) production

3.5 Osteoconductive potential

3.6 Statistical analysis

4. Results

4.1. Phase composition determination

4.1 Morphological Characterization

4.2 Specific surface area (BET)

4.3 Fourier—transform infrared spectroscopy (FTIR).

4.4 Thermal behavior

4.5 Analysis of the Ion Release Profile

5. Biological properties

5.1 In vitro cytocompatibility

5.2 ROS production

5.3 Osteoblast differentiation and proliferation

6. Discussion

7. Conclusions

Abbreviations

Declarations

References

Additional Declarations

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Journal Publication

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