TiO₂ Nanorod Arrays Modified Ti Substrates Promote The Adhesion,Proliferation And Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells

ObjectivesTitanium（Ti）and its alloys have been widely used as implants in orthopedic and oral implantology,due to their high strength and superior biocompatibility.However,failure and loss of implants because of inefficient osseointegration and microbial infection are still critical issues bothering researchers and clinicians.Recently,researches on surface modification of implants have been widely conducted to improve the osteogenic and antimicrobial bioactivities of implants.Nano-sized TiO₂ has a good application prospect in the field of biomedicine,due to its good biocompatibility and stable chemical and physical properties.TiO₂ nanostructures including nanoparticles and nanotubes have been widely investigated in the field of implant modification.In this study,assembled TiO₂ nanorod arrays（TNRs）were fabricated on the polished Ti samples.The adhesion,proliferation and osteogenic differentiation of human periodontal ligament stem cells（hPDLSCs）seeded on TNRs samples were evaluated to seek a novel method of implant modification.Methods:Assembled TNRs were fabricated on Ti samples using hydrothermal and sintering process.Polished Ti samples were used as control.Topography,crystalline structure,roughness and wettability of TNRs surface were examined using scanning electron microscope（SEM）,transmission electron microscope（TEM）,X-ray diffraction（XRD）,atomic force microscopy（AFM）and contact angle measurement,respectively.hPDLSCs were seeded onto TNRs and Ti samples.Initial adhesion of hPDLSCs on these two types of samples were observed by nuclear staining and count.Proliferation of hPDLSCs was examined by cell-counting kit-8（CCK-8）assay.Morphology of hPDLSCs on samples were observed under SEM and Confocal Laser Scanning Microscope（CLSM）.After osteogenic induction for 14d,cytoskeletal F-actin was evaluated by staining and quantitative real-time polymerase chain reaction（qRT-PCR）.After osteogenic induction for 7,14,21d,the osteogenic differentiation of hPDLSCs on these samples were evaluated by alkaline phosphatase（ALP）activity measurement,Alizarin Red S staining,cetylpyridinium chloride（CPC）colorimetry,qRT-PCR and western blot.Results:SEM and TEM images showed that the assembled TNRs were successfully synthesized on the surface of pure Ti.Each individual nanorod was about 1.μm long and about 100nm in diameter.The XRD patterns demonstrated that TNRs were typical rutile.Roughness,wettability and protein adsorption of TNRs were significantly higher than Ti（P<0.05）.The number of hPDLSCs adhered on TNRs substrates after 24 h was significantly higher than that on Ti substrates（P<0.05）.Moreover,hPDLSCs seeded on TNRs proliferated at a significantly higher rate than those on Ti（P<0.05）.hPDLSCs attached on TNRs samples exhibited more polygonal and stretched shape.After osteogenic induction for 14 d,F-actin expression of hPDLSCs on TNRs samples was siginificantly higher than that on Ti samples（P<0.05）.After osteogenic induction for 7,14 and 21 d,ALP activity,calcium deposition and mineralized nodules formation of hPDLSCs on TNRs samples were significantly promoted than that on Ti samples（P<0.05）.Moreover,the expression of ALP,runt related transcription factor 2（Runx2）and osteopontin（OPN）,measured by qRT-PCR and western blot was significantly up-regulated on TNRs samples when compared to Ti control（P<0.05）.Conclusions:TNRs fabricated onto Ti by hydrothermal reaction was biocompatible and significantly promoted the adhesion,proliferation and osteogenic differentiation of hPDLSCs.TNRs were expected to improve the osteogenic bioactivity of implants and become a novel surface modification method for implants.