Objective To test the surface properties and in vitro effects of a new sequential release system on MC3T3-E1 cells for improved osseointegration. Background BMP6-loaded anodized titanium coated with PDGF containing silk fibroin (SF) may improve osseointegration. Methods Titanium surfaces were electrochemically anodized, and SF layer was covered via electrospinning. Five experimental groups (unanodized Ti (Ti), anodized Ti (AnTi), anodized + BMP6-loaded Ti (AnTi-BMP6), anodized + BMP6 loaded + silk fibroin-coated Ti (AnTi-BMP6-SF), and anodized + BMP6-loaded + silk fibroin with PDGF-coated Ti (AnTi-BMP6-PDGF-SF)) were tested. After SEM characterization, contact angle analysis, and FTIR analysis, the amount of released PDGF and BMP6 was detected using ELISA. Cell proliferation (XTT), mineralization, and gene expression (RUNX2andALPL) were also evaluated. Results After successful anodization and loading of PDGF and BMP6, contact angle measurements showed hydrophobicity for TiO(2)and hydrophilicity for protein-adsorbed surfaces. In FTIR, protein-containing surfaces exhibited amide-I, amide-II, and amide-III bands at 1600 cm(-1)-1700 cm(-1), 1520 cm(-1)-1540 cm(-1), and 1220 cm(-1)-1300 cm(-1)spectrum levels with a significant peak in BMP6- and/or SF-loaded groups at 1100 cm(-1). PDGF release and BMP6 release were delayed, and relatively slower release was detected in SF-coated surfaces. Higher MC3T3-E1 proliferation and mineralization and lower gene expression ofRUNX2andALPLwere detected in AnTi-BMP6-PDGF-SF toward day 28. Conclusion The new system revealed a high potential for an improved early osseointegration period by means of a better factor release curve and contribution to the osteoblastic cell proliferation, mineralization, and associated gene expression.