Biocompatibility of barium-doped dicalcium phosphate dihydrate obtained via low-temperature synthesis for use in regenerative medicine

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BACKGROUND: Synthetic materials based on calcium phosphate compounds (CPCs) are increasingly used in modern regenerative medicine to stimulate bone tissue regeneration. AIM: The work aimed to assess key parameters of biocompatibility in vitro, including the content of acidic compartments and reactive oxygen species production, during the interaction of human macrophages with low-temperature-synthesized barium doped dicalcium phosphate dihydrate under normal and lipopolysaccharide (LPS)-induced inflammatory conditions.

METHODS: Morphology and qualitative and quantitative elemental composition of dicalcium phosphate dihydrate (DCPD) powder and its barium-doped form (DCPD-Ba) were evaluated using scanning electron microscopy, infrared spectroscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction. Cell viability, lysosomal content, and reactive oxygen species production were assessed by flow cytometry after co-culturing primary human macrophages with DCPD and DCPD-Ba under both normal and LPS-stimulated conditions.

RESULTS: Barium-doped DCPD samples were synthesized using a low-temperature method at Ba²⁺ concentrations of 1%, 5%, and 10% of the theoretically possible substitution level (theor.%). For each variant, the calculated actual substitution percentage amounted to 0.62, 1.43, and 6.43 atomic %, respectively. X-ray diffraction analysis confirmed complete transformation of the initial α-tricalcium phosphate into DCPD at all Ba2+ concentrations. The infrared spectroscopy data validated the conformity of DCPD structure to the reference standard across all Ba2+ doping levels. Doping with Ba2+ ions has been found to enhance the hydration activity of DCPD and cause deformation of its crystal structure. The results of in vitro studies indicate that substitution of Ca2+ with Ba2+ in the DCPD structure does not affect the cytotoxic properties of the material. Furthermore, DCPD-Ba did not suppress lysosomal biogenesis and promoted reactive oxygen species production in non-activated macrophages, whereas suppressing reactive oxygen species production under LPS-induced inflammatory conditions.

CONCLUSION: Thus, both DCPD and its Ba substituted variants represent promising candidates for incorporation into materials intended for regenerative medicine. The proposed low temperature synthesis of Ba2+-substituted DCPD is of considerable interest for the development of specialized osteoplastic CPC based materials. The most effective DCPD variant, with the highest Ca2+-to-Ba2+ substitution level (6.43 atomic %), demonstrated potential regulatory activity on activated macrophages (i.e., under inflammatory conditions). This property may be of critical importance for modulating the immune response and promoting effective osteointegration of the material in the recipient’s body.

Sobre autores

Polina Smirnova

Baikov Institute of Metallurgy and Materials Science of the Russian Academy of Sciences

Email: smirnova-imet@mail.ru
ORCID ID: 0000-0002-5437-7052
Código SPIN: 5022-2890
Rússia, Moscow

Anastasia Teterina

Baikov Institute of Metallurgy and Materials Science of the Russian Academy of Sciences

Email: teterina_imet@mail.ru
ORCID ID: 0009-0005-1405-2607
Código SPIN: 5514-8643

Cand. Sci. (Engineering)

Rússia, Moscow

Igor Smirnov

Baikov Institute of Metallurgy and Materials Science of the Russian Academy of Sciences

Email: baldyriz@gmail.com
ORCID ID: 0000-0003-3602-0276
Código SPIN: 3680-5330
Rússia, Moscow

Vladislav Minaychev

Baikov Institute of Metallurgy and Materials Science of the Russian Academy of Sciences; Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences

Email: vminaychev@gmail.com
ORCID ID: 0000-0002-8498-4566
Código SPIN: 9217-1374

Cand. Sci. (Biology)

Rússia, Moscow; Pushchino

Pavel Salynkin

Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences

Email: salynkin.p.s@gmail.com
ORCID ID: 0009-0002-0959-8072
Código SPIN: 2594-8099
Rússia, Pushchino

Margarita Kobyakova

Baikov Institute of Metallurgy and Materials Science of the Russian Academy of Sciences; Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences

Email: kobyakovami@gmail.com
ORCID ID: 0000-0002-6846-9994
Código SPIN: 5611-8437
Rússia, Moscow; Pushchino

Kira Pyatina

Baikov Institute of Metallurgy and Materials Science of the Russian Academy of Sciences; Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences

Email: kirapyatina01@gmail.com
ORCID ID: 0009-0003-0194-6922
Código SPIN: 2935-4432
Rússia, Moscow; Pushchino

Elena Meshcheriakova

Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences

Email: elena.mesh2311@gmail.com
ORCID ID: 0009-0001-6148-5211
Código SPIN: 6332-6772
Rússia, Pushchino

Irina Fadeeva

Baikov Institute of Metallurgy and Materials Science of the Russian Academy of Sciences; Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences

Autor responsável pela correspondência
Email: fadeeva.iteb@gmail.com
ORCID ID: 0000-0002-1709-9970
Código SPIN: 6475-1023

Cand. Sci. (Biology)

Rússia, Moscow; Pushchino

Sergey Barinov

Baikov Institute of Metallurgy and Materials Science of the Russian Academy of Sciences

Email: barinov_s@mail.ru
ORCID ID: 0000-0003-4544-2817
Código SPIN: 5876-1906
Scopus Author ID: 7004365385

Dr. Sci. (Engineering), Professor

Rússia, Moscow

Vladimir Komlev

Baikov Institute of Metallurgy and Materials Science of the Russian Academy of Sciences

Email: komlev@mail.ru
ORCID ID: 0000-0003-2068-7746
Código SPIN: 2668-0066

Dr. Sci. (Engineering), Professor

Rússia, Moscow

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2. Fig. 1. X-ray diffraction analysis of barium-doped dicalcium phosphate dihydrate powders at 1%, 5%, and 10% of the theoretically possible substitution level: DCPD, dicalcium phosphate dihydrate; DCPD-Ba, barium-substituted dicalcium phosphate dihydrate. X-axis: 2θ (degrees), Y-axis: intensity (relative units).

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3. Fig. 2. Infrared spectroscopy of barium-doped dicalcium phosphate dihydrate powders at 1%, 5%, and 10% of the theoretically possible substitution level: DCPD, dicalcium phosphate dihydrate; DCPD-Ba, barium-substituted dicalcium phosphate dihydrate.

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4. Fig. 3. Scanning electron microscopy of dicalcium phosphate dihydrate powders: a, original (undoped) dicalcium phosphate dihydrate; b, dicalcium phosphate dihydrate doped with 1% Ba²⁺ theoretical substitution level; c, dicalcium phosphate dihydrate doped with 5% Ba²⁺; d, dicalcium phosphate dihydrate doped with 10% Ba²⁺.

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5. Fig. 4. Cytotoxic effect of calcium phosphate compounds on human macrophages: DCPD, dicalcium phosphate dihydrate; DCPD-Ba, DCPD doped with Ba²⁺ at 1%, 5%, or 10% of the theoretical substitution level. a, percentage of viable cells relative to control after 72 h of incubation with various concentrations (10, 3, 1, 0.3, 0.1 mg/mL) of the tested calcium phosphate compounds; b, percentage of viable cells relative to control after 72 h of incubation with 10 mg/mL of DCPD or DCPD-Ba; c, percentage of viable cells relative to control after 72 h of incubation with 3 mg/mL of DCPD or DCPD-Ba; ** p < 0.01 compared with the control.

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6. Fig. 5. Acidic compartment content in human macrophages after incubation with calcium phosphate compounds: DCPD, dicalcium phosphate dihydrate; DCPD-Ba, DCPD doped with Ba²⁺ at 1%, 5%, or 10% of the theoretical substitution level. The Y-axis shows the mean fluorescence intensity of LysoTracker Green in cells, scale: ×106; ** p < 0.01 compared with the control group.

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7. Fig. 6. Production of reactive oxygen species in human macrophages after incubation with calcium phosphate compounds: DCPD, dicalcium phosphate dihydrate; DCPD-Ba, DCPD doped with Ba²⁺ at 1%, 5%, or 10% of the theoretical substitution level; LPS–, macrophages under standard (non-activated) culture conditions; LPS+, macrophages pre-incubated with lipopolysaccharide (activated); * p < 0.05, ** p < 0.01 compared with the control group without lipopolysaccharide; # p < 0.05, ## p < 0.01 compared with the control group with lipopolysaccharide.

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