Experimental Study Of Sustained Release Aspirin Liposome Enhanced Hydrogel In The Treatment Of Disc Degeneration Caused By Annulus Fibrosus Defect

Objective:Surgery was always the terminally choice for most patients who suffered intervertebral disc degeneration(1DD)with failed conservative treatment.However,operation focused on improve neural symptom and lacked the capacity of preventing reherniation and maintaining stabilization of mechanics.In this study,the sustained release and the repair function of aspirin liposome enhanced hydrogel were used to reduce postoperative disc inflammation and fill the annulus fibrosus defect to achieve a certain mechanical support,thereby postoperative disc degeneration was delayed.Methods:The ASP-Lip were prepared by thin film dispersion,and further incorporated into photo-cross-linkable GelMA.Then the material was photo-crosslinked as composite hydrogel material ASP-Lip+GelMA.Evaluating drug release in Liposome,structure in hydrogel,expansion and degradation in hydrogel,drug release in Liposome hydrogel.The nucleus pulposus cells of SD rats were co-cultured with the hydrogel and Liposome hydrogel material.The effects of the hydrogel material on nucleus pulposus cells and inflammatory factors influence were detected by CCK-8,ELISA,RT-PCR,Western-Blot and cellular immunofluorescence.L2-3,L3-4,L4-5 and L5-6 intervertebral discs of 20 New Zealand white rabbits were used to make defect models for comparative experiments.L2-3 was the normal control group,L3-4 was the vehicle group,L4-5 was the ASP-Lip+GelMA group,and L5-6 was the ASP+GelMA group.Postoperative mechanical experiments were performed,and X-Ray and MRI examinations were performed one and two weeks postoperatively.Tissue samples were got two weeks postoperatively,Hematoxylin-eosin staining(H&E)and safranin O-fast green staining were performed to observe histological morphology.Immunohistochemical stainings of IL-1?,IL-6,TNF-a,Collagen-II and MMP3 were performed to assess inflammation and degeneration of nucleus pulposus cells.Results:The ASP+GelMA expansion rate reached 617.93±78.55%within 100 h,but the ASP-Lip+GelMA basically maintained the expansion rate of 388.45±80.43%at 24 h(P<0.05).The total degradation time of ASP+GelMA was earlier than that of ASP-Lip+GelMA(21 d:28 d)(P<0.05).The sustained release of ASP-Lip+GelMA was significantly higher than that of ASP+GelMA.ASP-Lip+Gel groups had greater percentage of compression compared to the GelMA hydrogel,with 60.53±1.49%for ASP-Lip+Gel groups and 52.16±1.84%for ASP-GelMA.At the same percentage of compression,ASP-Lip+Gel suffered a resistance force of 48.29±2.69 N,which was significantly higher than 17.39±2.91 ASP-GelMA.In terms of compression modulus,the compression modulus of ASP-Lip+GelMA was 30.52±1.47kpa,higher than that of the ASP+GelMA group(19.91±1.16kpa)(P<0.05).ASP-Lip+GelMA can significantly maintain mechanical stability in terms of resistance,compression modulus and compression rate.The liposome and hydrogel materials themselves had no negative effect on the nucleus pulposus cells.ASP-Lip+GelMA could significantly inhibit the inflammatory reaction and reduce the loss of collagen-II and aggrecan.ASP-Lip+GelMA inhibited the expression of HMGB1 in the cytoplasm,while the expression in the nucleus was significantly increased.Postoperative results in vivo showed that ASP-Lip+GelMA can significantly maintain mechanical stability in terms of resistance,compression modulus and compression rate.DHI%in the vehicle group decreased significantly at the first week,from 100%to 78.10+2.21%(P<0.05),and continued to decrease two weeks after surgery(P<0.05).In the ASP-GelMA group and the ASP-Lip+GelMA group,the postoperative DHI%decreased from 100%to 80.10±3.01%and 89.30±2.25%respectively(P<0.05),and one week and two weeks after the operation,the ASP-GelMA group and the ASP-Lip+GelMA group showed smaller decreases in intervertebral space height than the vehicle group.ASP+GelMA and ASP-Lip+GelMA groups could better decrease the loss of collagen-II,aggrecan and water in intervertebral disc(P<0.05).Hematoxylin-eosin staining and safranin O-fast green staining showed that the normal and ordered intervertebral disc structure could be better maintained in the ASP-Lip+GelMA and ASP+GelMA groups compared with the vehicle group(P<0.05).Studies have shown that in histochemical staining,ASP-Lip+GelMA can significantly reduce the expression levels of IL-1?,IL-6,TNF-? and MMP3,and maintain the collagen-?.Conclusion:The results indicated that controlled release system based on ASP-Lip+GelMA seem significantly promising for improving degeneration and reduce reherniation or reoperation application in defect of nucleus pulposus after partly discectomy.This composite hydrogel material implied a promising strategy for the development of fillable material with preventing recurrent lumbar disc herniation after partly discectomy.