Lithium ion doped carbonated hydroxyapatite compositions: Synthesis, physicochemical characterisation and effect on osteogenic response in vitro

Abstract

Hydroxyapatite is a commonly researched biomaterial for bone regeneration applications. To augment performance, hydroxyapatite can be substituted with functional ions to promote repair. Here, co-substituted lithium ion (Li⁺) and carbonate ion hydroxyapatite compositions were synthesised by an aqueous precipitation method. The co-substitution of Li⁺ and CO₃^2- is a novel approach that accounts for charge balance, which has been ignored in the synthesis of Li doped calcium phosphates to date. Three compositions were synthesised: Li⁺-free (Li 0), low Li⁺ (Li 0.25), and high Li⁺ (Li 1). Synthesised samples were sintered as microporous discs (70-75 % theoretical sintered density) prior to being ground and fractionated to produce granules and powders, which were then characterised and evaluated in vitro. Physical and chemical characterisation demonstrated that lithium incorporation in Li 0.25 and Li 1 samples approached design levels (0.25 and 1 mol%), containing 0.253 and 0.881 mol% Li⁺ ions, respectively. The maximum CO₃^2- ion content was observed in the Li 1 sample, with ~8 wt% CO₃, with the carbonate ions located on both phosphate and hydroxyl sites in the crystal structure. Measurement of dissolution products following incubation experiments indicated a Li⁺ burst release profile in DMEM, with incubation of 30 mg/ml sample resulting in a Li⁺ ion concentration of approximately 140 mM after 24 h. For all compositions evaluated, sintered discs allowed for favourable attachment and proliferation of C2C12 cells, human osteoblast (hOB) cells, and human mesenchymal stem cells (hMSCs). An increase in alkaline phosphatase (ALP) activity with Li⁺ doping was demonstrated in C2C12 cells and hMSCs seeded onto sintered discs, whilst the inverse was observed in hOB cells. Furthermore, an increase in ALP activity was observed in C2C12 cells and hMSCs in response to dissolution products from Li 1 samples which related to Li⁺ release. Complementary experiments to further investigate the findings from hOB cells confirmed an osteogenic role of the surface topography of the discs. This research has shown successful synthesis of Li⁺ doped carbonated hydroxyapatite which demonstrated cytocompatibility and enhanced osteogenesis in vitro, compared to Li⁺-free controls.

Keywords: Bone; Carbonate; Hydroxyapatite; Lithium ions; Osteogenic differentiation.