The Experimental Study Of Effect Of The Peptide That Specifically Bind To Osteoblasts On Attachment And Migration Of Human Calvarial Osteoblasts In Vitro

The purified human calvarial osteoblasts were used as positive target cells,and the gingival fibroblasts were used as the control cells for adsorption.From the phage dopapeptide library,the peptides specifically binding to them were selected,namely,the osteoblast-specific identification polypeptides.Its amino acid sequence is:VLAVPWSEPGYL.The purpose of this study was to investigate the effect of the peptide on the adhesion and migration of human osteoblasts,and to determine the optimal concentration of the peptide for promoting migration and adhesion of human osteoblasts in vitro,and to provide experimental basis for the further study of the polypeptide.The whole study consists of four parts as follows.1.Cell adhesion experiment examine the proliferation of human calvarial osteoblastsHuman calvarial osteoblasts were routinely cultured which were divided into 6groups including experimental groups containing the ’peptide at various concentrations(10-4mol/L,10-5mol/L,10’6mol/L,10-1mol/L,10-8mol/L)and a control group.At corresponding time points(0.5h,1h,2h,4h,8h),the culture medium was discarded,and the number of cells adhered in the pores was calculated under the microscope by the stereological method.Each group took 6 regions and calculated the average(cell number/mm2)for statistical analysis.Results:the peptide that specifically binds to osteoblasts in the concentrations of 10-8 mol/L to 10-4mol/L could promote the adhesion of human calvarial osteoblasts in vitro,and the best concentration is 10-5mol/L2.Cell Scratch Repair Experiment and Transwell Migration Assay to examine the migrationof human calvarial osteoblasts.2.1 Scratch repair experiments:In vitro cultured human calvarial osteoblasts,with serum-free medium formulated into five concentration gradients(10-4,10-5,10-6,10-7,10-8mol/L)as Experimental group,serum-free medium without peptide as a control group,with 2ul pipette tip in the bottom of the culture medium covered with monolayer slowly vertical out of a straight trace,respectively,at 0,24,48 hours to observe the cells(= Area of pre-migration scratches-area of post-migration scratches),and statistical analysis was performed using the 24-hour scratch healing area as a statistical standard.Results:the peptide that specifically binds to osteoblasts in the concentrations of 10-8 mol/L to 10-4mol/L could promote the migration of human calvarial osteoblasts in vitro,and the best concentration is 10-4mol/L and 10-5mol/L.2.2 Transwell Migration Experiment:Human calvarial osteoblasts were cultured in vitro.The experiment was divided into six groups:blank control group,10-4mol/L group,10-5mol/L group,10-6mol/L group and 10-7mol/L group,10-8mol/L group.Transwell chamber(24-well microporous polycarbonate membrane,pore diameter 8.0um,membrane thickness 6.5mm),the upper room by adding 1 × 105 cells/ml containing 1%serum cell suspension 100ul,the lower chamber according to the experimental group added 600ul of culture medium containing 30%serum of different concentration of polypeptide,600ul of 30%serum culture medium containing polypeptide without peptide was added to the blank control group,and the cells were removed after 72h,fixed,stained,photographed and counted Experimental data for statistical analysis.Results:The peptide that specifically bind toosteoblasts in the concentrations of 10-8 mol/L to 10-4mol/L could promote the migration of human calvarial osteoblasts in vitro,and the best concentration is 10-5 mol/L.3.Transwell Migration Assay to examine the migration of human gingival fibroblasts and bone marrow stem cells.Transwell migration experiments:Human gingival fibroblasts and bone marrow stem cells were cultured in vitro.The experiment was divided into six groups:blank control group,10-4mol/L group,10-5mol/L group,10-6mol/L group,10-7mol/L group,10-8mol/L group.Transwell chamber(24-well microporous polycarbonate membrane,pore diameter 8.0um,membrane thickness 6.5mm),the upper room by adding 1×105 cells/ml containing 1%serum cell suspension 100ul,the lower chamber according to the experimental group added 600ul of culture medium containing 10%serum of different concentration of polypeptide and 600ul of culture medium containing 10%serum without peptide were added to the blank control group,and the cells were taken out after 24h and fixed,stained,photographed and counted Experimental data for statistical analysis.Results:The peptide that specifically binds to osteoblasts in the concentrations of 10’8 mol/L to 10-4mol/L could promote the migration of bone marrow stem cells in vitro,and could inhibit the migration of:Human gingival fibroblastsConculsion The peptide that specifically binds to osteoblasts can promote the migration and attachment of human calvarial osteoblasts in vitro.