| Background introduction:Laminectomy is one of the most common surgical methods in spinal surgery.Laminectomy is an essential step in the surgical treatment of intervertebral disc herniation,spinal canal stenosis,intraspinal tumor,lumbar spondylolisthesis and lumbar fracture.With the development of high-speed drill and micro instruments,laminectomy has become safer and more efficient.However,extradural scar adhesion after laminectomy often affects the surgical effect,resulting in lumbar surgery failure syndrome.After lumbar laminectomy,about 8%-40%of patients will have lumbar surgery failure syndrome,and 4%-9%of patients need a second operation.Therefore,how to prevent extradural scar adhesion after laminectomy is of great significance to improve the curative effect of spinal surgery,and it has also become a problem that spinal surgeons need to think about.In recent years,minimally invasive technology,drugs,biomaterial and non biomaterial barriers and other methods to prevent epidural adhesion are being deeply studied.In this study,the effect of cross-linked hyaluronic acid gel on the formation of epidural scar was studied by animal model and cell model.Part Ⅰ A study of cross-linked hyaluronic acid gel in the prevention of fibrosis and scar adhesion in lumbar epidural space after lumbar surgery in rabbitsObjective:To explore the safe dose of cross-linked hyaluronic acid gel(HA)in the prevention of scar adhesion in lumbar epidural space after lumbar surgery by motor function score(Basso,Beattie,Bresnahan;BBB)and somatosensory evoked potentials(SEPs).To study whether cross-linked HA can inhibit scar formation and reduce epidural adhesion in animals.Method:1.24 healthy male New Zealand white rabbits were randomly divided into one control group(n=6)and three experimental groups(n=6)after the dura mater was exposed with a dental drill(5 mm × 8 mm).All the experimental rabbits were tested with standardized preoperative motor function score.These was nothing covered on the dura surgical area in Group A.In Group B,C and D,0.5ml,1.0ml and 1.5ml cross-linked hyaluronic acid gel were covered on the dura surgical area respectively.And then closed the surgical incision.The latency of SEPs was measured after anesthesia,exposure of dura mater and covering with cross-linked hyaluronic acid gel or normal saline.And the BBB motor function rating scale was used to evaluate the motor function of rabbits on the 1st,3rd,5th and 7th day after operation.2.HE staining was used to observe the aggregation of fibroblasts in epidural scar tissue and Micro-CT was used to observe the effect of cross-linked HA on preventing epidural adhesion.RT-PCR was used to detect the genes that have been confirmed to be involved in the regulation of fibrosis or scar hyperplasia.Then the differentially expressed genes in RT-PCR were verified by Western blot analysis.Results:1.The latency of SEPs was measured for the first time after anesthesia,and there was no significant difference among the four groups(P>0.05).The latency of SEPs measured after exposure of dura mater for the second time was longer than that measured after the first anesthesia,but there was no significant difference(P>0.05).After covering cross-linked HA or normal saline,the latency of SEPs was measured for the third time.The results showed that the latency values of group A and group B were not significantly prolonged,and there was no significant difference between these two groups(P>0.05),while the latency values of group C and group D were prolonged.Compared with the latency values of SEPs measured for the first time after anesthesia,the difference was statistically significant(P<0.05).The motor function score showed that the scores of group A and group B were 21 points,which had no decrease compared with the score before operation;The motor function scores of group C and group D decreased in varying degrees on the first and third day after operation.However,on the fifth and seventh day after operation,the score returned to normal:there was no significant difference between the two groups compared with group A(P>0.05).2.The results of Micro-CT scan showed that the filling defect area of the epidural space in the experimental group was significantly smaller than that in the control group at the 4th and 6th week after operation(P<0.01).In addition,the quantitative analysis of fibroblasts showed that the number of fibroblasts in the control group was significantly higher than that in the experimental group at the second week after operation(P<0.05).The results of RT-PCR showed that the expression of TGF-β1 was down-regulated in the cross-linked HA group on the 3rd and 5th day after operation.In terms of collagen gene expression,COL1A1 expression was down-regulated on the 5th and 7th day after operation in cross-linked HA group.Then Western blot analysis was used to verify the differentially expressed genes screened in RT-PCR.The results also showed that the expression of TGF-β1 in the control group was significantly higher than that in the experimental group on the 3rd and 5th day after operation(P<0.05).In addition,the expression of COL1A1 in the control group was significantly higher than that in the experimental group on the 5th and 7th day after operation.The experimental results were consistent with the previous RT-PCR results.Conclusion:1.Through intraoperative SEPs monitoring and postoperative BBB function score,the safe coverage dose of cross-linked HA in the exposed dura area of 5 mm × 8 mm in rabbits was 0.5ml.2.In the rabbit model after laminectomy,cross-linked HA can reduce the epidural fibrosis by down-regulating the expression of TGF-β1 and COL1A1 mRNA,and then reducing the protein expression of TGF-β1 and COL1A1.Part Ⅱ A study of the mechanism of cross-linked hyaluronic acid gel to prevent epidural scar formation through cytokine secretionObjective:To investigate the inhibitory effect of cross-linked HA on inflammatory cytokines by macrophage model.To explore the feasible mechanism of cross-linked HA in preventing or reducing epidural scar formation from the perspective of cytokines.Method:The macrophage models of RAW264.7 mice were divided into control group(without any stimulation),lipopolysaccharide/endotoxin stimulation group(LPS group),cross-linked HA stimulation group(cross-linked HA group)and cross-linked HA+LPS stimulation group(cross-linked HA+LPS group).The supernatants were collected at different time points,and the concentrations of interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-8(IL-8)and tumor necrosis factor-α(TNF-α)in the supernatants of control group,LPS group,crosslinked HA group and cross-linked HA+LPS group were detected by enzyme-linked immunosorbent assay(ELISA).In vitro,macrophage model was used to detect the regulation and expression of TGF-β1 and COL1A1 by cross-linked HA by RT-PCR and Western blot.Results:Compared with the control group,LPS group showed that RAW264.7 cells initiated immune response immediately after being stimulated by LPS,and the secretion of IL-6,IL-1β,IL-8,and TNF-α was significantly higher than that in the control group.When RAW264.7 cells were stimulated by cross-linked HA,the results showed that there was no obvious immune response,and the secretion of inflammatory factors was almost the same as that of the control group.Compared with the LPS group,the inflammatory factor IL-6 secreted by the cross-linked HA+LPS group decreased significantly at the measured time point.There was no significant difference in other inflammatory factors IL-1β,IL-8,and TNF-α between LPS group and cross-linked HA+LPS group.The results of RT-PCR and Western blot showed that cross-linked HA could also down-regulate the expression of TGFβ1 and COL1A1 mRNA in vitro,and never decrease the protein expression of TGF-β1 and COL1A1.Conclusion:The application of cross-linked HA inhibited the production of proinflammatory cytokine IL-6 in macrophages stimulated by lipopolysaccharide,downregulated the expression of TGF-β1 and COL1A1 mRNA,and decreased the protein expression of TGF-β1 and COL1A1. |
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