Enhancement Of Cell Ingrowth,Proliferation,and Early Differentiation In Artificial Ligament Using Low-intensity Pulsed Ultrasound

Object:The purpose of this study was to use cell research in vitro to study the effects of low-intensity pulsed ultrasound(LIPUS)on the cell planted on the suiface of the artificial ligament ingrowth,proliferation,and early differentiation Methods: In vitro,MG63 cells grown on the surface of artificial ligament,were divided into control group(without ultrasonic stimulation)and experimental group(LIPUS stimulation),then the cells were evaluated for the differencesin cell activity(CCK-8 assay,confocal microscopy Calcein-AM/PI double staining,cell morphology(SEM)experiment,cell differentiation(alkaline)phosphatase content,osteogenic marker gene and protein expression)and apoptosis(Annexin V-FITC/PI staining).Results: Through SEM observation,the cell adhesion rate of the artificial ligament membrane on the ultrasound stimulation group is high,and the membrane substrate is bare.Meanwhile,the number of cells in the unstimulated group is small,and there is a large area of basal exposure.At the same time,from the observation of cell morphology,ultrasonic stimulation group film surface cells aggregation growth trend,and no stimulation group surface cells scattered in the growth of cells connected closely,furthermore,ultrasound stimulation group cells were round,pseudopods number,but the no stimulation group surface were slightly smaller and less.Confocal microscopy showed that a large number of living cells could be seen on the surface of the artificial ligament membrane in the ultrasonic stimulation group,and the number of viable cells on the surface of the unstimulated group was relatively small.In the number of dead cells,only a small amount of dead cells could be seen on the surface of the artificial ligaments in the stimulus group,and the number of dead cells was less than that of the unstimulated group.CCK-8 results suggest that from the overall trend,the OD value of the two groups from the first day to the fifth day culture showed an upward trend,but from the view of each time point.The cell viability was significantly higher than that in non stimulated group.In terms of cell differentiation,alkaline phosphatase two group surface cells showed an upward trend,from the view of each time point,alkaline phosphatase stimulating cell group artificial ligament of fabric surface secretion was significantly higher than the untreated group;at the same time,according to the q-PCR and Western results of blot,ultrasound stimulation group consisting of bone related gene and protein expression were significantly higher than that of no stimulation group.The results of flow cytometry showed that the apoptosis of the surface of the artificial ligaments on the ultrasonic stimulation group was significantly lower than that in the unstimulated group.Conclusion: These results suggested that ultrasound treatment with low-intensity acoustic energy facilitated the cellular ingrowth and enhanced the proliferation and early differentiation of osteoblasts in artificial ligaments...