Osteoporosis, a bone disease caused by the decreased mass and impaired structure of bone, is becoming an increasing threat to the health of the elderly population. To treat and prevent this kind of suffering, understanding of the bone formation and regeneration is of significant importance, during which bone cell-biomaterial interaction plays a critical role, especially osteoblasts. In this work, we report an in situ investigation of osteoblast proliferation, migration and differentiation on a series of substrates with variable surface chemistry as a result of coating fabrication. Primary investigation indicated that cells migrated faster on sulfonated polystyrene (SPS)-coated MirrIR slides than uncoated slides but grew slower. Cell elongation results were well consistent with the migration data. To explain these differences, hydrophilicity of SPS-coated and uncoated slides was measured, which turned out that SPS coating made the slides more hydrophobic. However, to successfully explain the results one challenge is that multiple discrepancies co-exist between SPS-coated and uncoated MirrIR slides, excluding surface chemistry, surface roughness and surface charge, which requires future work focusing on realization of univariate parameter at one time. Final understanding of effects of individual parameter listed above on osteoblast proliferation, migration and differentiation is hopeful to help design new implantable materials for clinical surgery.