| Objective: To investigate the preparation method of HA/CS-TGF-beta 1 composite coating by physical,chemical and biological modification on titanium surfaces and obtain the specific physical morphology,chemical composition and biochemical modification on titanium surface,so as to make it have a biological function.Study on its effect on the adhesion and proliferation of osteoblasts and evaluate its cell toxicity and effect of bone cell characteristics of gene expression.To provide a reliable theoretical basis and experimental basis for improving the biocompatibility of titanium implants by surface modification method,and to promote the clinical application of the composite coatings.Methods: HA / CS-TGF-β 1composite coatings were prepared on titanium surfaces by physical,chemical and biological modification.To make the 10mm*10mm*1mm titanium plate polished clean and micro arc oxidation treatment,and put them into the dopamine solution for 18 h.After dry in room temperature,Dripping the HA/CS solution,The experiment adopts the negative pressure treatment 10 min,dry 24 hours,The titanium discs were randomly divided into five groups as follows:A、B、C、D、E group,respectively,0.4g gelatin microspheres with 20 ml 0.1g/ml CS solution mixing 20 min,joined 0.12 gNHS after adding dissolution of 0.08 gEDC in ice bath,then immediately added TGF-1 protein.The added protein concentration were 15ng/ml,30ng/ml/,60ng/ml/,75ng/ml,250ng/ml.After cross linking reaction of 12 h,drying,scanning electron microscope(SEM),and X ray diffraction(XRD),infrared spectroscopy(FTIR)and other instruments were used to analyze its chemical composition and the surface topography.Separation of rat osteoblasts and cultured in primary and passage,with a concentration of 100μl/ hold were cultured on the disks of each group.At the time point of l d,3d and 7d,each hole with CCK-8 reagent solution 20 ul,37 DEG incubated for 2.5 h after each hole to take 110 ul liquid into the 96 well plates,the OD values was measured by CCK8 to evaluate the proliferation.osteoblasts with a concentration of 1 x 1 03/cm2 were cultured on the disks of each group.At the time point of 6h and 3d,inverted fluorescence microscope observe the cells.The cells were cultured with each group of titanium plate.At the time point of l d,2d,3d,5d and 7d,sampling on osteoblast mRNA,for purification,transcription,amplification.The ALP,Runx2,TGF-electrophoresis,beta 1 bands ALP,Runx2,TGF-beta 1 bands will be in electrophoresis.Results: HA / CS-TGF-β 1composite coatings were successfully prepared,the SEM show that:experimental group titanium plate surface are basically consistent with the small pore and carrying TGF beta 1 protein gelatin microspheres were spherical,uniform particle size,visible on the surface of protein adsorption.Through the analysis of X-ray diffraction,found that titanium matrix peak was almost obliterated,the main chemical composition is Ti02(anatase).Its contact angle is almost zero,which is super hydrophilic.With the "burst release" effect at the beginning of drug release,and then drug release slowly,to the release of the drug release of 7D(58.4 + 1.8)%,14dTGF-protein beta 1 release concentration of(80.1 + 1.9)%.groups:As the culture time increased,the activity of proliferation increased.At l d,there is no significant difference among groups.At 3 d,the number of osteoblasts were significantly increased,group B and C is higher than other groups,At 7 d,B,C cell proliferation experiment group was significantly higher than other groups(P<0.05),E group cell density is less than the other groups(P<0.05).Osteoblasts were seeded in the experimental group and the control group titanium plate for 6h and 3d,after DAPI dyed into bone cell nuclei,we found that: number of cells with no obvious difference at 6h,in 3D,in each group were significantly proliferation,especially in experimental group B and C,the cell number of E group is relatively small,number of cells in all experimental groups significantly more than that in the control group,indicating that composite coating for cell adhesion and the early proliferation has a significant role in promoting.In a certain concentration of TGF-beta 1,the expression of ALP and Runx2 was increased with time,B and C groups are obvious,while the expression of TGF-beta 1 basically unchanged.Conclusion:1、the drug release system of HA/CS-TGF-beta 1 composite coating is stable and maintained for a long time,It can be a long time for the organization to provide TGF-beta 1.2、The contact angle of HA/CS-TGF-beta 1 composite coating is almost zero,which is super hydrophilic.the hydrophilic surface is conducive to cell adhesion and proliferation.3 、 HA/CS-TGF-beta 1 composite coating can promote osteoblast proliferation and the release of protein concentration in a certain range,the effect is particularly obvious4、HA/CS-TGF-beta 1 composite coating can promote osteoblast marker gene expression,thereby promoting osteoblast proliferation. |
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