Effect Of Chimeric Peptide MinTBP-1-PRGDN On Adhesion, Proliferation And Differentiation Of Osteoblasts On Titanium Surfaces In Vitro

Titanium (Ti) has been widely used as an embedding biomaterial in dental implants, artificial joints and so on, owing to its good biocompatibility, osseointegration, resistance to corrosion and low allergenicity. The key point of successful implant is the achievement of fine stability between Ti and bone tissue. Various kinds of natural or artificial peptides and proteins have been widely investigated to promote osseointegration. The RGD (Arg-Gly-Asp) sequence, which can be found in most extracellular matrix (ECM) proteins, including some mineralized-related proteins, is the most effective and most often employed peptide sequence for improving cell adhesion on biomaterial surfaces. Stable immobilization of peptides on the surface of biomaterials is essential to promote strong cell adhesion. RGD peptides commonly covalently bind to the surface of biomaterials via functional moieties such as hydroxyl, amino, and carboxyl radicals. However, many problems may arise in these coupling methods:besides the complex treatment procedures, the coupling reagent may be cytotoxic or the active group may be deactivated quickly by hydrolysis. A new peptide aptamer, named TBP-1 (RKLPDAPGMHTW) which is isolated from a linear 12-mer peptide phage library, has been proved to have the character of specifically interacting with the surface of Ti. Its N-terminal, RKLPDA (minTBP-1), is sufficient for Ti binding. Considering its specific and strong binding ability, minTBP-1 has been used in the form of combination with other motifs, which endowed the chimeric peptides with specific binding ability to Ti. In this study, we synthesized a chimeric peptide with double motifs containing a RGD and a minTBP-1 sequence, and investigated its binding ability to Ti and its effect to osteoblast on attachment, spreading, proliferation, differentiation and mineralization ability as well.Part One:The interaction between minTBP-1-PRGDN and titaniumObject To explore the affinities of minTBP-1-PRGDN, minTBP-1and PRGDN to Ti at different concentrations; the different affinities of three peprtides to unoxidized and oxidized Ti; the influence of FITC labling on peptide affinity.Methods Experiment one:Ti disks were wet-polished, washed and sterilized sequentially, and incubated overnight with three peptides separativly at four different concentrations of 1μg/ml,10μg/ml,100μg/ml and 1000μg/ml, then washed with ddH2O to remove unbound peptides. Experiment two:Ti disks were wet-polished, washed and sterilized sequentially. Half numbers of disks were oxidized with 30% HNO3 and the others were not oxidized. The elements on the surfaces of oxidized and unoxidized Ti disks were detected by XPS. Then all disks were incubated overnight with three peptides separativly at concentration of 100μg/ml, and then washed with ddH2O to remove unbound peptides. Experiment three:Ti disks were wet-polished, washed, oxidized and sterilized sequentially, and then incubated overnight with the combination of unlabeled and FITC-labeled peptides in the proportion of 0:1,10:1, and 100:1, washed with ddH2O to remove unbound peptides. Photos of coated disks were taken by fluorescence microscope and analyzed by Image-Pro(?) Plus software package by measuring the average number of fluorescent pixels per disk.Results Experiment one:The numbers of binding prptides rose with the concentrations increasing, and the binding of three peptides were close to saturation at 100μg/ml. Experiment two:The propotion of oxygen on oxidized disks increased. The affinity of minTBP-1-PRGDN and minTBP-1 to oxidized Ti disks increased while that of PRGDN dramatically decreased. Experiment three:With the increasing proportion of unlabeled peptide mixing into labeled peptide, fluorescent pixels in three groups all decreased, the FITC labling did not affect the peptide.binding ability.Part Two:The effect of minTBP-1-PRGDN on osteoblast adhensionObject To explore the effects of different Ti coatings with minTBP-1-PRGDN, minTBP-land PRGDN on osteoblast attachment, spreading and the expression of adhension-relative gene. Methods Ti disks were wet-polished, washed, oxidized and sterilized sequentially, and then incubated overnight with three kinds of unlabled peptides separativly, washed with ddH2O to remove unbound peptides. Osteoblast were seeded on Ti disks and adhered for some time(1h in experiment 4 and 5,24h in experiment 6), and then non-adhesive cells were washed with PBS. Experiment four:The quantification of adhesive cells was colorimetry using Alamar Blue. Experiment five:The morphous of adhered adhesive cells were stained with phalloidin-FITC and taken photos with fluorescence microscope. And then the extent of cell spreading on Ti disks was measured quantitatively by measuring the cell areas with Image-Pro? Plus software package. Experiment six:The adhension-relative gene integrinβ1 and cadherin-11 were detected by RT-PCR.Results Experiment four:The number of attached cells was most on minTBP-1-PRGDN pre-coated disks and least on PRGDN pre-coated disks. Experiment five:Cells adhering on minTBP-1-PRGDN pre-coated disks exhibited active spreading and mainly oblate or round shape, lacking in pseudopodia. Cells in PRGDN pre-coated group were almost cuboidal with a few pseudopodias and had the least spreading area. Experiment six:The adhension-relative gene Integrinβ1 and Cadherin-11 were expressed most obviously in PRGDN group.Part Three:The effect of minTBP-1-PRGDN on osteoblat proliferationObject To explore the effects of different Ti coatings with minTBP-1-PRGDN, minTBP-1and PRGDN on osteoblast proliferation, including cell numbers and the expression of proliferation-relative gene.Methods Experiment seven:MC3T3-E1 osteoblasts proliferated on different pre-coated disks for 24h,48h and 72h, and then cell numbers were evaluated by MTT assay, and cell shape and density were observed with metallurgical microscope after Giemsa's staining. Experiment eight:The proliferation-relative gene collagen Iα1 and cyclin Dl were detected by RT-PCR.Results Experiment seven:Cell numbers in all groups were increasing with the time in four groups. At 24h, cell number of minTBP-1-PRGDN group was the maximum and significant difference was found by comparing with control group. Osteoblasts presented fusiform shape and lined according with the orientation of Ti disk burnishing. Experiment eight:The expression of cyclin D1 gene were increasing with the time in all groups except control group. At 72h, the gene expression of cyclin Dl was most conspicuous. For the expression of collagen Iα1 gene, PRGDN group was the most at 24h, and control group was the least at 48h, and minTBP-1 group was the most at 72h.Part Four:The effect of minTBP-1-PRGDN on osteoblat differentiation and functionObject To explore the effects of different Ti coatings with minTBP-1-PRGDN. minTBP-1and PRGDN on osteoblast differentiation and function, including ALP activity, expression of differentiation-relative gene, and formation of mineralized nodules.Methods Experiment nine:Osteoblasts were incubated, ALP activity was determined as the rate of p-nitrophenol release from p-nitrophenylphosphate substrate at day 3,7,10,14, 21 and 28. Experiment ten:The differentiation-relative gene were detected by RT-PCR, including ALP, OPN, BSP and OC. Experiment eleven:After being incubated with differentiation medium for 28d, osteoblasts were stained with Alizarin red-S for displaying mineralized nodules. And then nodules were taken photos with metallurgical microscope and their numbers and size were measured quantitatively with Image-Pro? Plus software package.Results Experiment nine:ALP activity increased slowly from day 3 to 10, and increased acceleratively from day 14 and reached the peak value at day 28. Experiment ten: OPN,OC,BSP and ALP genes in minTBP-1-PRGDN group all increased firstly and dropped afterwards. However, the four genes in minTBP-1 group increased sustainedly with time. In control group, OPN,BSP and ALP genes increased sustainedly, but OC gene decreased firstly and raised then. In PRGDN group, OPN,OC and ALP genes decreased firstly and raised then, but BSP increased firstly and dropped afterwards. The expression of four genes highestly in PRGDN group on day 7, and in minTBP-1-PRGDN group on day 14. Experiment eleven:The average area of mineralized nodules was the biggest in minTBP-1-PRGDN group and the smallest in minTBP-1-group. The numbers of nodules were the most in PRGDN group and the least in minTBP-1-group.