Surface Submicron Structure Of Poly(ether-ether-ketone)Materials And Its Effect On Osteogenic Differentiation Of Mesenchymal Stem Cells

Objective To modified the surface bioactivity of Polyetheretherketone(PEEK)through nitrification and sulfonation.And to observe the surface morphology and research the biological properties of the modified PEEK.Methods 1.Different surface morphology of PEEK was formed by sulfonation and nitrification.Then the mixtures with residual acid,sulfur and nitrogen on the surface of PEEK were removed by water bath at different temperatures.Five groups were designed in the present experiment,including control group(PEEK),sulfonationand nitrification with 25℃ water bath(25NP),sulfonation and nitrification with 100℃ water bath(100NP),sulfonation with 25℃ water bath(25SP)and sulfonation with 100℃ water bath(100SP).PEEK was characterized by field emission scanning electron microscopy,atomic force microscopy,energy dispersive spectrometer,contact angle measuring instrument and Fourier transform infrared spectrometer.2.In vitro,DAPI staining,vinculin staining,phalloidin staining,CCK-8 assay,alkaline phosphatase(ALP)activity staining,alizarin red(ARS)qualitative and quantitative detection were used to detect the initial adhesion,cell morphology,proliferation,mineralization and other cell behavior of bone marrow mesenchyme stem cells(BMSCs)on PEEK,respectively.3.In the molecular experiments,the expression of ALP,OPN,COL-1,BMP-2 and Runx2 was detected by q RT-PCR and WB to research the osteogenesis of BMSCs on PEEK with different surface morphology.Results 1.According to the results of surface detection,the surface of PEEK group was smooth;NP group was with monolayer submicron pore structure and sulfonic and nitro groups were introduced into the main chain of the molecule;SP groups was with three-dimensional reticular structure and sulfonic groups were introduced into the main chain of the molecule.AFM results showed that the surface roughness of PEEK group was smaller than other groups.NP group had well-regulated surface morphology and relative roughness.SP group had irregular surface morphology and was with the highest roughness.The content of sulfur and nitrogen of the surface of PEEK decreased significantly after the treatment of high temperature hydrothermal treatment,and the surface morphology and hydrophobicity of PEEK were not affected by hydrothermal treatment at different temperatures.PEEK group had the strongest surface hydrophilic ability.And NP group was weaker and SP group was the weakest(P<0.005).2.In vitro,the early adhesion of BMSCs on the surface of SP and NP group was more than that of PEEK group(P<0.05).Early adhesion of BMSCs of 100 SP was more than 100 NP group,but it had no Statistical difference.The results of morphological observation showed that part of BMSCs in group PEEK was not completely spread out,and the number of pseudo foot was less.In group NP and SP,BMSCs were spread well,polygonal or star shaped,and pseudo foot was rich,but the surface cell adhesion of 100 NP group was closer than that of 100 SP group.At 2h,confocal microscopy showed that cells on PEEK group showed incomplete cytoskeleton and plaques.Other groups had developed complete microfilaments and stress fibers,and plaque proteins were relatively clear.At 4h,the cytoskeletons of all groups were well developed,and the binuclear cells were observed in group 100 NP and 100 SP.Cells in PEEK group were smaller in volume and less spread.At 24 h,the number of cells in the NP and the SP groups was significantly increased than PEEK group and was similar to osteoblast.CCK-8 results showed that the proliferation of cells in NP and SP groups increased significantly compared with PEEK group(P<0.05)at 4,7 and 14 days,and the proliferation ability of 100 SP group was superior to that of 100 NP group(P<0.05)at 7 days.Alkaline phosphatase results suggested that,at 7 and 14 days,NP and SP group of cells on the surface showed material matrix fusion,dark staining and showed a blue black,PEEK group showed weak staining.Quantitative ALP results suggested that the expression of NP group compared with SP group increased significantly(P<0.05)at 7 days.The results of alizarin red staining showed that the mineralized nodules of the NP and SP groups were obvious and larger compared with that of PEEK group.The quantitative results of ECM were in accordance with the staining results.3.The q RT-PCR results showed,At 7d,treatment group compared with PEEK group: the expression of ALP,RUNX2 were significantly increased(P<0.05);100NP group compared with 100 SP group,the expression of ALP and RUNX2 were significantly increased;At 14 d,treatment group compared with PEEK group: the expression of ALP and RUNX2 increased slightly,OPN,Col-1 expression increased.The results of Western blot suggest that the expression levels of RUNX2,OPN and BMP-2 in group PEEK were all lower than those in treatment group.On the 7 day,the expression of RUNX2 and BMP-2 increased in group 100 NP compared with other groups.On the 14 day,the expression levels of OPN and BMP-2 in 100 NP group were higher than those in other groups.Conclusions After sulfonation and nitrification,the single layer submicron pore structure was obtained on the surface of PEEK.Sulfonation on the surface of PEEK obtained a three-dimensional network structure.High temperature water bath can improve the bioactivity of BMSCs,including early adhesion,proliferation and differentiation mineralization.Monolayer submicron pore structure can promote the early expression of the gene and protein related to bone occurrence.