Effect Of Mechanical Stimulation On Matrix Metabolism Of Annulus Fibrosus-Derived Stem Cells And Repair Of The Annulus Fibrosus Defect

Objective: To compare metabolism gene expression of rabbit annulus fibrosus-derived stem cells(AFSCs) under different mechanical tensile strength; To evaluate efficacy of AFSCs undergoing mechanical stimulation for the repair of rabbit annulus fibrosus.Methods: We gained annulus fibrosus of New Zealand white rabbits(1.5kg) and isolated and cultured their AFSCs; The cultured AFSCs were divided into 3 groups which underwent mechanical stretch stimulation of 0%,2%, 5%, 12%(0.5Hz,4h/d,3d). The changes of shape and anabolism gene expression(Collage-I,Collage-II,Aggrecan) of AFSCs were explored with cytoskeleton staining and RT-qPCR technology in vitro. Then determined the most appropriate intensity of mechanical stimulation based on these results. Eighteen healthy New Zealand white rabbits were randomly divided into 3 groups(2-week group, 4-week group, 6-week group) according to the culture cycle. The model of annulus fibrosus defect was established in the 3 groups: nothing was given in the Blank group(L3-4); Gelfoam group(L4-5) were only stuffed with gelatin sponge; Stretched AFSCs group(L5-6) were stuffed with gelatin sponge and given AFSCs undergoing mechanical stimulation; AFSCs group(L6-7) were stuffed with gelatin sponge and given untreated AFSCs. Eighteen sacrificed animals took X-ray(anteroposterior and lateral view) and MRI at 2 weeks, 4 weeks, 6 weeks respectively. Compare efficacy of repair of different groups based on the hydration and area of nucleus pulposus, intervetebral disc height index(DHI) and pathology of repairing sites(H-E staining and Masson staining).Results: The results of cytoskeleton staining of AFSCs under 0%, 2%, 5%, 12%(0.5Hz, 4h, 3d) mechanical tensile strength showed there were no significant changes in cell morphology between these groups, but cytoskeleton staining of AFSCs which underwent 2% and 12% mechanical stimulation were more blurring. In addition, gene expression of matrix metabolism(Collage-I, Collage-II, Aggrecan) of AFSCs undergoing 2% and 12% tensile stimulation decreased lower tensile strength compared with control group. The anabolism gene expression of AFSCs undergoing 5% tensile stimulation increased significantly compared with other groups. We considered that mechanical stimulation(5% of intensity, 5Hz of frequency, 4 hours each day, 3 days) was the most appropriate; The results of MRI showed the degenerative process can be deferred by Stretched AFSCs group and AFSCs group; Hydration and area of nucleus pulposus showed a decreased tendency in Blank group and Gelfoam group at 2 weeks after surgery. Compared with it, data of Stretched AFSCs group and AFSCs group were higher than other groups in each period. However, there was no significant difference between two AFSCs groups; DHI of Blank group and Gelfoam group had a decreased tendency. DHI of Stretched AFSCs and AFSCs groups decreased after 2 weeks, then rising slowly, which had a significant difference with other groups. Results of pathology showed there was intact nucleus pulposus, well-defined annulus fibrosus and clear boundary in Stretched AFSCs group and AFSCs group at 2 weeks and 4 weeks. Defect sites were stuffed with fibroblasts and chondrocytes. Compared with the previous, intervertebral disc degenerated and nucleus pulposus decreased significantly in Stretched AFSCs group, AFSCs group at 6 weeks.Conclusion: 5% mechanical stimulation can promote matrix metabolism gene expression of AFSCs effectively. AFSCs transplanted into defect sits can defer the degeneration and promote repair. However, due to the lack of statistical support, it was necessary to make more profound research in the efficacy of AFSCs undergoing mechanical stimulation.