| Low back pain and sciatica are among the most common medical problems in mordern society, affecting many people at some time during their lives, which are caused by lumbar disc herniation (LDH) most commonly. Operation is a main method to treat LDH and laminectomy is a routine procedure in the operation of symptomatic LDH. After laminectomy, the symptoms such as back or leg pain didn't relieve or recured after relief, then it would cause operation failure and lead to failed back surgery syndrome (FBSS). Epidural scar is considered one of the most common causes that could cause FBSS. Just like wound healing in the other parts of the body, epidural fibrosis after laminectomy is a natural consequence of normal wound healing. When the epidural scar lead to nerve root tethering or dura adhesion, the symptoms such as back or leg pain may recur. Some studies indicated that patients having extensive epidural scar have been shown to be 3.2 times more likely to experience recurrent radicular pain than those patients with less extensive epidural scarring. Most recurrent symptoms caused by epidural scar need repeated operations, which are considerably hampered by the presence of scar tissue. Moreover, after excision of the epidural scar, the fibrous tissue may recur, leading to failure of surgical outcome. Therefore, it is necessary to investigate the causes and the prevention of epidural scar formation.There is widespread concern about the study of materials which can prevent epidural scar. Various materials have been used in attempts to minimize epidural fibrosis with varying results, but none have provided optimal results. In the recent years, PLGA [poly (lactic-co-glycolic acid)] was widely concerned for its good biocompatibility and biodegradability which can be regulated,and has been ratified to use in medical field by FDA. Among the medicines that can inhibite fibroblast, interferon has inhibitory effect on skin fibroblast. So we think that interferon should have the inhibitory effect on epidural fibroblast, too. Making composite membrane with PLGA and some scar inhibitors, such as interferon and chitosan, and using the composite membrane to prevent epidural scar, we estimate the scar adhesion could be prevented for the barrier ability of PLGA and the effect of scar inhibitors on fibroblasts.The study observed the effects of recombinant human interferonα-2b (rhIFNα-2b) on the biological behavior of fibroblasts derived from epidural scar preliminarily, and investigated the ability of IFN/PLGA composite membrane,chitosan/PLGA composite membrane,PLGA membrane and Sodium hyaluronate,autogenous free fat to prevent epidural scar adhesion after laminectomy in rabbits.Part 1: L3 laminectomies were performed in rabbits, 0.5×1.2 cm2 defect areas were made and the dura maters were exposed. 3-4 weeks later the epidural scars were obtained. Using the technique of cell culture, the fibroblasts derived from epidural scar cultured in vitro were observed by means of cell counting and MTT assay, then the effects of rhIFNα-2b on the biological behavior of epidural scar fibroblasts were analysed. Part 2: L2 and L4 laminectomies were performed in rabbits, 0.5×1.2 cm2 defect areas were made and the dura maters were exposed respectively. Then different procedure was carried out randomly: 1, Nonimplant control group: nothing on epidurals; 2, Autogenous free fat graft group: the exposed duras were covered with subcutaneous free fat 0.5×0.5×1.5cm3; 3, Sodium hyaluronate group: dripping sodium hyaluronate 1 ml on dura maters; 4, PLGA membrane,chitosan/PLGA composite membrane,IFN/PLGA composite membrane groups: The exposed duras were covered with one of the three membranes with suitable size; 20 rabbits in each group. The animals were killed at 2,4,6,8 weeks postoperatively, and observed as follows: 1, Gross observation: the degree of scar formation and adhesion to dura in L2 laminectomy site was observed and evaluated according to the Robertson score criteria. 2, Histology observation: the whole L4 vertebrae including paraspinal muscles and tissues were obtained, the specimens were fixed and decalcificated, the slices were made and hematoxylin and eosin (HE) staining and Masson's trichrome staining were performed. Then the histological evaluation was made. 3, Electron microscope observation: epidural scars were obtained 4 weeks after operation and the fibroblast ultrastructure was observed by transmission electron microscope (TEM). 4, Immunohistochemistry observation: L2 spinal cords were obtained, the transverse sections of spinal cords were made, which were examined by immunohistochemical staining with Fos. The expression of c-fos was observed and the Fos-LI cells were counted. 5, Revers Transcription Polymerase Chain Reaction (RT-PCR): the expression of TGF-β1 in the epidural scars were analysed with RT-PCR technology.Results of Part 1: The body of fibroblast was plump, the cell's boundary was clear in fusiform shape and the cell connected by 2-3 processes in control group. With the increase of concentration of IFNα-2b in experimental group, the density of fibroblasts became low and cells distributed loosely. The bodies of cells became small and some turned round, the processes of cells became shortened. The results of cell count and MTT assay showed that compared with the control group, the fibroblasts derived from epidural scar in experimental group had changes on inhibition of cell growth and proliferation, and the inhibitory action of concentration at≥4000U/ml to the proliferation of fibroblasts were obvious. With the increase of concentration of rhIFNα-2b, the inhibitory action was stronger. Though the fibroblasts could be inhibited by rhIFNα-2b, the number of fibroblasts still increased. Results of Part 2: 1, Gross observation: with time going on, scars formed in each group. Widespread scars formed in control group, and adhesions to the duras were tight. The scars and the adhesions in fat group and PLGA group were less than that in control group, but no significant difference between fat and PLGA group. In early stage, the scars in sodium hyaluronate group were less than that in fat and PLGA groups obviously, while in later period the difference between them was not significant, but every time the results of sodium hyaluronate group were better than that of control group. Little scar and scarce or slight adhesion to dura in chitosan /PLGA and IFN/PLGA composite membrane groups could be found, There was significant difference between the two groups and other groups. 2, Histology observation: scar tissue formed in every time in every group. In control group, the scar tissue was wide and the adhesion to dura was tight. The scar tissue,collagen and adhesion in fat group and PLGA group were less than that in control group, but no significant difference between fat group and PLGA group. The free fat decreasing and absorption gradually could be found. Less scar tissue,collagen and adhesion to dura in sodium hyaluronate group than that in control group were found, in later period there was no significant difference between sodium hyaluronate group and fat,PLGA groups. The results of chitosan/PLGA and IFN/PLGA composite membrane groups were best. Compared with other groups, significant difference could be obtained. 3, Electron microscope observation: at 4 weeks postoperatively. There were many fibroblasts and collagen fibers in control group. More plentiful rough endoplasmatic reticulums were found, which indicated the status of fibroblast was good. The difference between fat group,PLGA group and control group was not significant. In sodium hyaluronate group,chitosan /PLGA group and IFN/PLGA group, few fibroblasts with few rough endoplasmatic reticulum and mitochondrion were found, which indicated the status of fibroblast was not so good and the proliferation was inactive. 4, Immunohistochemisty observation: there was expression of c-fos in spinal cord, which was mainly in superficial layer of spinal cord dorsal horn and the expression increased with time going on. The data of Fos-LI cell count showed that there was significant difference between experimental groups and control group. Later period there was significant difference between chitosan /PLGA group,IFN/PLGA group and other groups. 5, RT-PCR: there was much expression of TGF-β1 in control group and 8 weeks later the expression was less than before. At every time there was significant difference between chitosan /PLGA group,IFN/PLGA group and control group.According to the results above, the conclusions can be obtained: rhIFNα-2b can suppress growth and proliferation of fibroblasts derived from epidural scar. rhIFNα-2b is a negative modulation factor of fibroblast but it can't cease the proliferation entirely. PLGA has good biocompatibility and biodegradability. Autogenous free fat and PLGA membrane have good mechanical barrier effect and can prevent epidural scar adhesion, but no significant difference between the two groups. In early stage the effects of sodium hyaluronate on preventing epidural scar and adhesion are obvious, while in later period the preventing effects are not so better than before, but still better than control group. Chitosan /PLGA composite membrane and IFN/PLGA composite membrane can reduce epidural scar formation and decrease the degree of adhesion to dura obviously, which are better than other groups, but no significant difference between the two groups.The study observed the effects of rhIFNα-2b on the biological behavior of fibroblasts derived from epidural scar preliminarily, and found rhIFNα-2b has the positive inhibitory effect on epidural fibroblasts. The PLGA membrane and the composite membranes with PLGA and IFN or chitosan were used to prevent epidural scar adhesion in rabbits and the composite membranes were proved more effective, but no significant difference between the two composite membranes. Chitosan /PLGA composite membrane and IFN/PLGA composite membrane are effective materials to prevent epidural scar adhesion after laminectomy.
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