Effects of surface roughening and calcite coating of titanium on cell growth and differentiation

Medical pure titanium (Ti) exhibits excellent mechanical properties and chemical stability in clinical use, but its initial osteointegration period is often postponed due to the bioinert nature of the Ti surface. Roughening and bioactive material coating of Ti implant surfaces are considered effective to enhance the bioactivity of Ti implants. In this study, we evaluated the effects of surface roughening and calcite (CaCO₃) coating of Ti substrates on osteoblastic cell differentiation and growth. We roughened the Ti substrate surface by acid etching, followed by coating with calcite by thermal decomposition of Ca(NO₃)₂ to CaO followed by thermal carbonation of CaO to CaCO₃. The surface topography of roughened Ti substrates (rough Ti) fluctuated, and the arithmetic average of surface roughness (Ra) was 2.2 µm. The rough topography was retained even after calcite coating of rough Ti, and the calcite-coated Ti (calcite-Ti) has an Ra of 2.0 µm. The tensile adhesive and shear adhesive strengths between calcite coating and Ti surface in calcite–Ti were 56.6 ± 16.1 and 10.1 ± 1.39 MPa, respectively. The biological properties and response of calcite–Ti were evaluated in vitro using a pre-osteoblastic cell line (MC3T3-E1). Observation of cell morphology by scanning electron microscopy and immunofluorescence staining revealed that MC3T3-E1 cells attached favorably to the surface with polygonal and filopodial extensions on calcite–Ti. The combination of roughening and calcite coating of the Ti substrate surface significantly increased cell proliferation at 1, 3, and 7 days of incubation. Furthermore, the relative alkaline phosphatase activity of calcite–Ti was higher than that of untreated Ti substrates (smooth Ti) and rough Ti after incubation for 7 days. Thus, the combined surface roughening and calcite coating of Ti substrates promoted MC3T3-E1 differentiation, whereas roughening alone was not effective.