| Background：Titanium is a widely used biological material for boneand dental implants due to its excellent mechanical and biological properties,but long-term clinical observations and studies suggest that the boneintegrational ability of this kind of materials is relatively poor[1]. How toimprove the biological properties of titanium, increase its compatibility withthe surrounding tissue, promote and accelerate the bone integration hasbecome focuses of many studies[2-4]. Many scholars argue that the materialsurface morphology and structure is one of the most important factors thataffect cell attachment and growth Compared with plane titanium, TiO2nanotube is more advantageous to induce mesenchymal stem cells (MSCs)osteogeneic differentiation due to its high aspect ratio and orderly onedimensional internal structure[5]. But there were conflicts about the effectsof different diameter TiO2nanotubes on cell adhesion, proliferation anddifferentiation[6-8]. MSCs are multipotent adult stem cells[9-10]. How toeffectively induce it osteogeneic differentiation has become a hot topic inbone tissue engineering[11-17]. Scholars believe that different cultures have different influences on the osteogeneic differentiation of MSCs[13], andregular medium with L-Ascorbic acid, β-Glycerophosphate anddexamethasone is a classic method for in vitro inducing MSCs osteoblastdifferentiation[16]. TiO2nanotubes have been applied in solar cells andhydrogen production by water photoelectrolysis and especially used asimplant biomaterial. And TiO2nanotubes can also serve as carriers fordrugs[18-21].Objective：1. To investigate the effects of different diameter TiO2nanotubes prepared by anodic oxidationon on the adhesion, proliferation andosteogenic differentiation of MSCs derived from Sprague Dawley rats.2. Toinvestigate the osteogenic differentiation of MSCs derived from SpragueDawley rats on TiO2nanotubes under different inducing conditions.3. Tofabricate DEX-loaded TiO2nanotubes and evaluate its function on adhesion,proliferation and osteogenic differentiation of MSCs.Methods：The isolated MSCs were characterized by osteogenic andadipogenic differentiation and flow cytometry. TiO2nanotubes of30,70nmand100nm were fabricated by anodization with different voltage.1. Thetopography and the hydrophilicity of the nanotubes were respectivelyaccessed with scanning electron microscope(SEM) and contact angle test.MTT colorimetric assay was applied to evaluate the cell adhesion andproliferation on different substrates. Cell morphologies were observed byconfocal laser scanning microscopy (CLSM). The activity of alkaline phosphatase and mineralization of MSCs on plane titanium and30nm,70nm,100nm TiO2nanotubes were also detected during osteogenic differentiation.The expression of Runx2and OSX were analyzed by real-time PCR after14days osteogenic differentiation.2. Four osteogenic differentiation methodswere approached: Regular culture medium (Control group), regular mediumwith L-Ascorbic acid and β-Glycerophosphate, regular medium withL-Ascorbic acid, β-Glycerophosphate and1α,25-Dihydroxyvitamin D3andregular medium with L-Ascorbic acid, β-Glycerophosphate anddexamethasone. ALP activity of all groups were analyzed after3,7, and14days culture, respectively. Calcium deposition was detected after21daysculture. Real-time PCR was applied to quantitate the expression ofosteoblast related gene Runx2and OSX after14days culture.3.Dexamethasone (DEX) was then loaded onto30,70nm and100nm diameterTNT arrays and multilayered coatings of gelatin/chitosan were subsequentlyperformed by layer by layer self-assembly (LBL) technique, confirmed bySEM, Contact angle measurement and X-Ray Fluorescence Spectrometer(XRF). Release studies of DEX from TNT arrays with multilayered coatingswere performed with a UV spectrophotometer. Cell morphologies of MSCscultured on DEX-loaded TiO2nanotubes for24hours were imaged byCLSM and SEM. MTT colorimetric assay was applied to evaluate the celladhesion and proliferation on the substrates. The activity of alkalinephosphatase (after3,7and14days culture), mineralization (after21days culture) and Runx2and OSX gene expression (after7,14and21daysculture) of MSCs on plane titanium and30nm,70nm,100nm TiO2nanotubes were also detected during osteogenic differentiation.Results： MSCs had been successfully induced osteogenic andadipogenic differentiation, and CD29positive cells were99.65%, CD44positive cells were99.81%. CD45positive cells were1.42%. Nanotubes ofdifferent diameter had been generated and met our requirement observed bySEM.1. TiO2nanotubes of70nm diameter were more hydrophilic（P<0.05）while cell adhesion and proliferation were also much greater compared withother substrates. The activity of alkaline phosphatase in cells cultured on30nm TiO2nanotubes was much higher than other groups after7daysosteogenetic induction（P<0.05）, and the mineralization (after21days) aswell as Runx2and OSX gene expression (after14days) were also higher onthe30nm group（P<0.05）.2. MSCs cultured on the same surface structuredemonstrated higher ALP activity (F=338.542, p=0.000), calciumdeposition (F=417.012, p=0.000) as well as Runx2(F=14.419, p=0.000) andOSX (F=42.011, p=0.000) gene expression in the L-Ascorbic acid+β-Glycerophosphate+dexamethasone group than in the others. Thedifference was statistically significant. Within the same culture condition,ALP activity (F=53.170, p=0.000), calcium deposition (F=264.268, p=0.000)and Runx2(F=3.196, p=0.037) and OSX (F=5.895, p=0.003) geneexpression were much higher on the surface structure of30nm diameter TiO2 nanotubes. The difference was also statistically significant. Culturecondition and material structure have interactions (ALP activity (F=6.322,p=0.000), calcium deposition (F=33.330, p=0.000) and gene expression ofOSX (F=2.825, p=0.015)), in which L-Ascorbic acid+β-Glycerophosphate+dexamethasone culture and30nm diameter TiO2nanotubes is the bestcombination.3. Release studies of DEX from TNT arrays with multilayeredcoatings in a14-day immersion study demonstrated an initial burst releasefollowed by sustained release. Cell morphologies visualized by confocallaser scanning microscopy (CLSM) and SEM indicated well spreading andadherence on DEX-loaded TiO2nanotubes. MSCs on DEX-loaded TiO2nanotubes demonstrated higher adherence and proliferation compared withsmooth titanium according to MTT assay results, Meanwhlie, MSCs onDEX-loaded TiO2nanotubes showed higer ALP and mineralization levels,as well as the gene expression of Runx2and Osterix (OSX) compared withsmooth titanium(p<0.05).Conclusion：1. TiO2nanotubes with different diameter have differentinfluences on MSCs, compared with plane titanium. TiO2nanotubes withdiameter of70nm are more advantageous for cell adhesion and proliferation,while TiO2nanotubes with diameter of30nm facilitate osteogenicdifferentiation.2. The osteogenic differentiation of MSCs can be influencedby both the structure of material surface and chemical stimulation. On thesame surface structure, MSCs in the L-Ascorbic acid+β-Glycerophosphate +dexamethasone culture group have greater potential of osteogenicdifferentiation than in the others. Under the same chemical stimulation,MSCs on30nm diameter TiO2nanotubes were more apt to differentiatingtowards osteoblasts than the way on other surface structures. Thecombination condition of L-Ascorbic acid+β-Glycerophosphate+dexamethasone culture and30nm diameter TiO2nanotubes is mostconductive to the osteogenic differentiation of MSCs.3.The fabricated DEX-loaded TiO2nanotubes demonstrated controlled release capacibility andcould promote adhesion, proliferation and osteogenic differentiation ofMSCs. |
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