Ionic substitutions have been proposed as a tool to control the functional behavior of synthetic hydroxyapatite (HA), particularly for Bone Tissue Engineering (BTE) applications. The effect of simultaneous substitution of different levels of carbonate (CO 3 ) and silicon (Si) ions in the HA lattice was investigated. Furthermore, human bone marrow-derived mesenchymal stem cells (hMSCs) were cultured on multi-substituted HA (SiCHA) to determine if biomimetic chemical compositions were osteoconductive. Of the four different compositions investigates, SiCHA-1 (0.58wt% Si) and SiCHA-2 (0.45wt% Si) showed missing bands for CO 3 and Si using FTIR analysis, indicating competition for occupation of the phosphate site in the HA lattice. 500°C was considered the most favourable calcination temperature as: (i) the powders produced possessed a similar amount of CO 3 (2-8wt%) and Si (<1.0wt%) as present in native bone; and (ii) there was a minimal loss of CO 3 and Si from the HA structure to the surroundings during calcination. Higher Si content in SiCHA-1 led to lower cell viability and at most hindered proliferation, but no toxicity effect occurred.While, lower Si content in SiCHA-2 showed the highest ALP/DNA ratio after 21 days culture with hMSCs, indicating that the powder may stimulate osteogenic behaviour to a greater extent than other powders.