| Peripheral nerve injury is one of the most common diseases in clinical surgery.At present,clinical autologous nerve graft is the "gold standard" for peripheral nerve defect repair,but its clinical application is limited due to its limited source,secondary nerve function of sacrificed donor area and other factors,so artificial nerve provides a new idea for clinical repair,and has become a research focus of peripheral nerve repair.In recent years,with the development of tissue engineering technology,many materials have been applied in the preparation of artificial nerves.Silk is a common tissue engineering material with high toughness and good biocompatibility.Previous studies of our team have shown that the microchannel artificial nerve conduit constructed with silk fibers has a significant effect on guiding and promoting the growth of regenerated nerve fibers,but it is difficult to degrade in vivo.Subsequent studies have shown that cellular phagocytosis in the inflammatory response is one of the main factors for degradation of silk fibers in vivo,and moderate inflammatory response also helps promote the repair of injured nerves on the early stage of nerve injury repair.Studies show that after peripheral nerve injury,the inflammatory response is triggered immediately,and the injury site and the full-length nerve fibers at the distal end undergo Wallerian degeneration.In the process,macrophages engulf degenerated tissue debris and remove obstacles for nerve regeneration,Schwann cells proliferate and activate to form the Büngner band,and secrete neurotrophic factors to promote axon regeneration.LPS(lipopolysaccharide)is the main component of the cell wall of Gram-negative bacteria.As a typical immune response activator,it can effectively stimulate the activation of macrophages and glial cells by activating the corresponding TLRs signaling pathway,and induce the Inflammation in vivo.Based on the above reasons,this study constructed a silk fiber microchannel composite artificial nerve conduit containing LPS.On the one hand,through the continuous release of LPS within a certain period of time,it induces a moderate inflammatory response,accelerates the recruitment of inflammatory cells and removal of myelin sheaths and axon fragments afters during Waller's degeneration after sciatic nerve injury in rats.On the other hand,it promotes the phagocytosis of silk fibers by macrophages,thereby further promoting the degradation of silk fibers in vivo;aiming to shorten WD's time after nerve injury,accelerate the speed of nerve regeneration and improve the repair effect of nerve conduit from an immunological point of view,provide new ideas for the in-depth study of artificial nerve conduit and the auxiliary treatment of peripheral nerves.In order to explore the effect of the dose of LPS on inflammatory cells,this study first used different doses of LPS to inject tail veins into rats,and used a full-automatic hematology analyzer to perform routine blood tests;then calcium alginate gel was used as a slow-release carrier,LPS-loaded silk fiber microchannel composite artificial nerve conduit were prepared and tested for their role in repairing peripheral nerve defects.For this reason,a total of three artificial nerve conduits were designed,respectively four graft schemes were carried out,namely: autograft group(control),silk fibers group,gel group and LPS group.At the corresponding time points after transplantation,sciatic nerve function index(SFI)detection,HE staining and immunofluorescence staining were performed to evaluate the effect of three artificial nerve conduits in repairing nerve defects and the activity of inflammatory cells in the repair process.Finally,real-time PCR was used to test the expression of lysosome-related genes and inflammatory cytokines during injury repair,and to explore the changes in inflammation and silk degradation activity in peripheral nerve injury repair.The results showed that:1.The results of routine blood analysis in rats show that both low-dose(1 ?g/kg)LPS and medium-dose(100 ?g/kg)LPS can significantly induce the proliferation of leukocytes in rat blood,while high-dose(10 mg/kg)LPS causes a decrease in the number of leukocytes,suggesting that a certain dose of LPS can induce a moderate inflammation in rats.2.Artificial nerve in vitro test:(1)The results of in vitro sustained release of LPS in neural conduits show that the release rate of LPS is fast during the first 7 days,and then the release rate gradually tends to be gentle,and the cumulative release amount on the 21 st day is 2444.2 EU,the release is basically over.The above results show that the LPS-loaded silk fiber microchannel composite artificial nerve conduit can slowly release LPS to a certain extent,and has a good sustained release effect.(2)The artificial nerve conduit material was co-cultured with Schwann cells.The results of CCK8 measurement showed that the three kinds of artificial nerves had no obvious toxicity to Schwann cells and showed good biocompatibility.3.Evaluation of the effect of artificial nerve repair:(1)HE staining observation showed that the nerve fibers regeneration status of the grafts in each group was good.At the 12 th week after surgery,the silk fibers in the three kinds of artificial nerve conduits showed different degrees of degradation.Among them,the number of remaining silk fibers per unit area in the LPS group was the smallest,indicating that the addition of LPS promoted the degradation of silk fibers in vivo.(2)Immunofluorescence observation showed that in the first week after surgery,the proximal and distal nerve tissues of the 4 groups were infiltrated by a large number of macrophages.Among them,the number of macrophages in the LPS group was significantly higher than that of other graft groups;Demyelination was observed both proximal and distal nerve tissues.Compared with the proximal end,the degree of demyelination was higher in the distal end.In each groups,the remaining axons and myelin fragments were the least in the LPS group.A large number of Schwann cells and regenerated axons were detected in all groups of graft at the 12 th week after surgery.Schwann cells and regenerated axons were the most in the autograft group,and the number of Schwann cells and regenerated axons in the remaining 3 groups is more to less in order: LPS group,gel group,silk fibers group.(3)The results of the sciatic nerve function index(SFI)at 2th,4th,and 12 th weeks after surgery showed that the SFI of each group gradually recovered over time,and the LPS group was significantly better than other artificial nerve groups,and the repair effect was closest to the autograft group.4.Real-time PCR results showed that the proinflammatory cytokines IL-6 mRNA and TNF-? mRNA began to be up-regulated significantly on the third day after nerve injury,and the anti-inflammatory cytokine IL-10 mRNA began to be up-regulatedon significantly on the second week after nerve injury,slightly later than pro-inflammatory cytokines;the relative expression of IL-6 mRNA in LPS group was significantly higher than that in other groups within 2 weeks of injury.Lysosome-related gene mRNA of each graft group was up-regulated significantly in different periods after surgery,and Ctsd mRNA was up-regulated significantly on 4th weeks.Hip1 r mRNA began to be up-regulated significantly on first week,while Plat mRNA began to be up-regulated significantly on the third day.In summary,the experimental results show that the lipopolysaccharide / silk fiber / PLA composite artificial nerve conduit constructed in this study can induce a moderate inflammatory response,accelerate the clearance of axon and myelin fragments during the WD's process on the early stage of nerve injury,and promote the sciatic nerve structure and functional recovery;in addition,the addition of LPS can also promote the degradation of silk fibers in vivo. |
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