Mesenchymal stem cell secretome discovery in response to a brushite-coated titanium alloy: highlighted a specific signature of factors involved in bone healing.

Increased use of titanium (Ti) and its alloys in implant manufacture is due to their biocompatibility and mechanical properties. However, their biological inertness must be considered. Surface modifications are essential for accelerating osteointegration and bone healing. Herein, calcium phosphate (brushite, CaHPO.2HO) was deposited on an additive-manufactured Ti6Al4V alloy substrate by electrochemical deposition technique. X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy confirmed the presence of brushite on the Ti6Al4V substrate. Scanning electron microscopy (SEM) and cross-section micrograph observations confirmed the homogeneity of the coating's coverage, composed of microcrystals approximately 1-3 μm thick and 10 μm long. EDXS analysis revealed pure brushite stoichiometric values, and tensile adhesion tests demonstrated good adhesion to the substrate. The coating demonstrated a rapid dissolution in MES (pH 5.5) and HEPES (pH 7.4) buffer solutions. Human mesenchymal stem cells (hMSCs) cultured with brushite-coated Ti alloy in osteogenic medium showed normal proliferation and increased biomineralization with SP7 mRNA upregulation. To explore how Brushite improved bone healing, we performed liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics analysis of hMSCs secretome from co-cultures with Ti alloys, for 7 and 14 days. Brushite promoted secretion of osteogenic and bone matrix factors. Factors involved in blood clotting (e.g. FII, FV, FX), mitochondrial biogenesis, energy metabolism (e.g. mitochondrial ATP synthase), lipid metabolism (e.g. apolipoproteins) and cellular stress response (e.g. heat shock proteins) were enriched. Increased specific chromatin-related proteins suggest chromatin's role in enhancing bone regeneration. Secretome profiling showed the unique role of brushite Ti alloys in bone regeneration, encouraging further study.