| 【Background】Orthopedic percutaneous implants play an important role in the treatment of severe compound injuries and complex fractures in the body,and the key to the success of its clinical application lies in percutaneous sealing.If the seal is not good,however,it can cause bacteria invasion,needle infection,material loosening and other complications.【Objective】In addition to the direct integration of the material-skin interface,the factors affecting the percutaneous sealing also include the dynamic mechanical environment with the somatic movement.Therefore,this project combines the groove modification of the material surface and dynamic mechanics to build the best percutaneous sealing method.We aims to find an ideal balance point for regulating sealing,which can not only promote rapid sealing,but also maintain the elasticity of surrounding soft tissues,improve the application experience of percutaneous implants and reduce complications.【Methods】Part Ⅰ1.We entrusted Beijing Mi G Lab to complete the preparation of groove pattern by micro-nano processing technology.The specific groove size parameters are: the groove width is fixed at 30 μm,the ridge width is 1 μm,5 μm,10 μm or 30 μm respectively,defined as class I chips;the ridge width is fixed at 5 μm,the groove width is 5 μm,10 μm,20 μm or 30 μm respectively,and the groove depth is 10 μm,defined as class II chips.2.The contact angle meter was used to measure the water contact angle of the samples.3.BMSCs of 2-week-old SD rats were extracted in primary culture,and the third generation cells were identified by flow cytometry and subsequent experiments.4.P38-MAPK inhibitor SB203580 was added to the culture medium of BMSCs inoculated on the material,and intervention experiments were conducted.5.We observed and semi-quantified cell morphology,adhesion,fluorescence intensity ofα-SMA,CTGF,Col-I,and p38 MAPK,and the intervention effect of SB203580 on stem cell differentiation and ECM by using immunofluorescence in whole and scanning electron microscopy(SEM)in part.Part Ⅱ1.We entrust Xi’an Institute of Optics and precision machinery of Chinese Academy of Sciences to process the groove pattern on the flat titanium sheet.The groove size is: 60 μm groove width,80 μm ridge width,and 10 μm groove depth.2.The contact angle meter was used to measure the water contact angle of the samples.3.NIH-3T3 cell line and BMSCs extracted from 2-week-old mice primary extraction were used in this experiment.The third generation BMSCs were used for follow-up experiment.4.The live / dead assay was used to determine the biocompatibility of planar titanium sheets and cell viability.5.We commissioned Northwestern Polytechnical University to make a small experimental oscillator,which can provide cells with reciprocating fluid shear.The cyclic reciprocating fluid frequencies were set to 0.2 Hz,0.6 Hz,and 1.0 Hz,respectively,and static culture was used as a control.6.SEM,immunofluorescence,PCR and immunoblotting were used to observe and semi-quantify and quantify changes in cell morphology,adhesion,α-SMA and Col-I expression under different frequencies of fluid.7.Scratch test was used to detect the effects of fluid shear stress on cell migration and the role of groove structure in the above effects.【Results】Part Ⅰ1.Flow cytometry showed that CD29 and CD90 positive cells accounted for the vast majority,suggesting that the primary cells extracted were BMSCs.2.Changes in ridge width or groove width have no significant effect on hydrophilicity.3.Compared with the other groups on the first type of chips,the 5 μm and 10 μm ridge width groups were arranged in an orderly manner.The cell area was relatively small,and the cell polarity was obvious.As the ridge width increased,the cells gradually migrated to the ridge.There was no significant difference in cell alignment,area,polarity and distribution of cells in the groove width group of 10 μm to 30 μm on the second type of chip,but the former was slightly more polar than the latter.4.The ridge width of 5 μm was the smallest dimension that the cell extended across.If it was less than this width,most cells could not be crossed and were arranged in the plane area along the edge of the groove-plane boundary.5.F-actin and α-SMA were not sensitive to changes in ridge width,but changed proportionally with groove width,the latter was more sensitive than the former the width of groove.The fluorescence intensity of vinculin,CTGF and Col-I was the strongest in the5 μm ridge width group and the 30 μm groove width group,and the FA area was the largest in the 5 μm ridge width group.The change in groove width did not affect the FA area.The FA number of a single cell was proportional to the cell area.6.The expression of phosphorylated P38 was highest in the 5 μm ridge width group and the 30 μm groove width group compared with other groups.SB203580 could inhibit the expression of phosphorylated P38 in BMSCs on most groove structures.7.SB203580 could inhibit the expression of α-SMA,CTGF and Col-I in BMSCs on most groove structures.Part Ⅱ1.SEM and atomic force microscope showed that the flat titanium sheet had a smooth structure,and the static water contact angle manifested that the titanium sheet was weak hydrophilicity.2.The results of the live / dead assay showed that the cell compatibility of the flat titanium sheet was similar to that of the glass slide,indicating that the titanium sheet used had good cell compatibility.3.The volume fluctuation in the culture caused by the evaporation of liquid and the subsequent addition of medium in the fluid shear system did not affect the maximum and average shear stress,that was,it did not affect the accuracy and repeatability of the results.4.On a flat titanium sheet,compared with other frequency groups,fibroblasts were most aligned in the direction of 0.6 Hz shear force,with the most obvious polarity.Moreover,there was no obvious correlation between cell angle and polarity.5.Immunofluorescence,gene and protein immunoblotting results of vinculin on a flat titanium film showed that vinculin expression and the average size of FA appeared the highest value in the 0.6 Hz group.6.On flat titanium sheets,the fluorescence intensity,gene and protein expression of Col-I and α-SMA were most sensitive to 1.0 Hz stress.7.Under static conditions,the groove structure could promote the alignment of the cell direction and reduce the cell area compared with the flat titanium sheet,but did not affect the polarity of the cell.8.Relatively static culture,0.6 Hz fluid shear force promoted the alignment,polarization,migration,adhesion and number distribution ratio of fibroblasts on the groove,but did not affect the cell area on the groove.9.Relative to the flat titanium sheet,the fluid shear force of 0.6 Hz enhanced the polarization and migration of fibroblasts on the grooves and reduce the cell area,but did not affect the cell alignment and adhesion.10.On a flat titanium sheet,relatively static culture,fluid shear force of 1.0 Hz increased the expression of Col-I of stem cells,but did not affect the expression of α-SMA.11.Relatively static culture,1.0 Hz fluid shear force enhanced Col-I and α-SMA protein expression of stem cells on the groove.12.Compared with the flat titanium sheet,the fluid shear force of 1.0 Hz promoted the protein expression of Col-I and α-SMA of the stem cells on the groove.【Conclusion】1.The main factor determining the tissue fibrosis was the change of the groove width.The degree of fibrosis would increase with the widening of the groove width in the range of 30μm,and the collagen arrangement tended to be disordered.Changing the ridge width did not affect the stability of stem cell differentiation into fibroblasts,which was conducive to tissue regeneration.The orderly and dense degree of secreted ECM with the ridge width of at least 5 μm was inversely proportional to the ridge width.The above results suggested that the narrow ridge width facilitated percutaneous integration in the design of the percutaneous part of the external fixation.But the size of the ditch needed to be determined in further animal experiments,and a narrow groove width did not necessarily mean an advantage.Because a certain degree of myofibroblast differentiation is essential for the early repair of skin and soft tissue,wide grooves can accelerate the differentiated myofibroblasts to formation of collagen around the implant,which has a positive effect on preventing infection and accelerating sealing.Only the groove width that achieves a balance between repair speed and tissue texture is the optimal groove width for optimizing percutaneous bioseal.2.0.6 Hz stress had the greatest influence on the alignment,polarity,migration and adhesion of fibroblasts on plane by increasing the expression of reoriented actin and vinculin;whereas 1.0 Hz stress promoted differentiation of fibroblasts into myofibroblasts by increasing Col-I and α-SMA expression.Interestingly,under the given frequency stress,the groove structure strengthened the above characteristics of fibroblasts beyond adhesion,and promoted differentiation of BMSCs into myofibroblasts.The above results indicate that 0.6 Hz may improve the implant-tissue sealing,while 1.0 Hz stress probably causes the disordered fiber deposition around implants. |
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