| Titanium and its alloys have been widely used as endosseous dental implant materialsin the past decade because of their mechanical strength, stability, and goodbiocompatibility. Implant surfaces play an important role in affecting osseointegration.Many studies have focused on the osseointegration process, and it is now described at boththe histological and cellular levels. Previous work has shown that surface characteristicssuch as roughness, wettability and surface structure significantly influence celldifferentiation, and consequently, osseointegration. Bone marrow mesenchymal stem cells(BMMSCs) are multipotent stem cells and represent a particularly attractive source fortissue engineering. BMMSCs are the first cells to colonize the biomaterial surface afterimplantation and are associated with osteogenic differentiation capacity. BMMSCs havebeen utilized in numerous studies as a source of osteogenic cells for bone repair.MethodsTitanium disks (15mm in diameter and1mm thick) of grade2commercially pure titanium were prepared by machining. These disks were immersed in NaOHpredominantly dissolves only in water with a concentration of2.5,5,7.5,10and12.5M.Unprocessed titanium disks were used as controls. The surface of the specimens wasexamined by scanning electron microscopy (SEM) and atomic force microscopy (AFM).Contact angle measurements were carried out by video contact angle measurement systemmodel VSA2500XE. Energy Dispersive X-ray Detector (EDX) was used to analyze thecomponents of all specimens. The protein absorption of specimens was determined bycommercial Protein Assay Kit. The rat BMMSCs were obtained from the femurs of8-week-old Sprague–Dawley rats. Cell adhesion was assessed by the method of stainingand counting with fluorescence microscope. Cell proliferation was measured using theCellTiter-Blue Cell Viability Assay. The cell morphology was observed using SEM.Alkaline phosphatase (ALP) activity and ECM mineralization were assessed usingcommercial kits. The sandwich enzyme immunoassay used in this study was specific forrat osteocalcin. The expression of several osteogenesis-related genes was assessed usingReal time-PCR.Results1.Similar porous network structures were formed by the alkali treatment. Thehigh-magnification SEM images show details of the TNS self-organization into a porousnetwork structure. The dimension of the porous network structures became smaller from2.5to10M NaOH. However, the porous network structures were nonhomogeneous with12.5M NaOH. AFM also showed similar appearances, with uniform roughness.Thecontact angles of the alkali-treated specimens gradually diminished from40°to6°withincreasing alkali concentrations, except12.5M NaOH. The alkali treated specimensshowed that components of the surface are the main of Ti element and micro Na and Sielements. With the increase of the alkali concentration, Na element was also had littleincreased, but no significant differences.2.More protein was adsorbed onto the alkali-treated specimens than the controlspecimens, which increased with the alkali concentrations, except12.5M NaOH.3.The cell numbers on alkali-treated specimens clearly increased with alkali concentration after incubation for30min,1h and3h. However, cell adhesion on thespecimens treated with12.5M NaOH was lower than that on the surfaces treated with10M NaOH.4.For short time culture of30min,1h and3h, the BMMSCs showed a similarelliptical shape; however, the filopodia appeared noticeably different. The quantity andlength of filopodia increased with alkali concentration. When cultured for a long time (3days), the BMMSCs showed markedly different shapes related to the specimen topography.BMMSCs in the control group displayed a spindle shape and developed less distinctlamellipodia, which was indicative of undifferentiated BMMSCs. In contrast, the cells onexperiment groups’ surfaces displayed a stellate cell shape with well-developed, highlyspread lamellipodia.5.In1and2weeks, ALP activity and the ALP staining results was showing obviousdifferences in the ALP activity among different specimens. In3and4weeks, the ECMmineralization levels and osteocalcin production in experiment groups’ were significantlyhigher than in the control group.6.The expression levels of osteogenesis-related genes including, BSP, ON, RUNX2and COL-1were assessed by quantitative RT-PCR. The similar topographies explored inthis study induced different gene expression levels at3and7days. Generally,alkali-treated specimens induced higher mRNA levels than the controls.ConclusionNano-network topography on titanium surface can be fabricated by alkali treatment,of which the characteristics such as the dimension of the nano-network structures,roughness, wettability and protein adsorption are closely related to the alkali concentration.The nano-network structures induced by alkali etching markedly enhanced BMMSCsfunctions of cell adhesion and osteogenesis-related gene expression, in addition other cellbehaviors such as proliferation, ALP activity, and ECM deposition and mineralizationswere also significantly improved. These effects were most pronounced when theconcentration of NaOH was10M. In particular, these results are experimental basis forthe next animal experiments and provide theoretical support for the future clinical application. |
