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

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.