| Peripheral nerve injury is one of most common diseases.Nerve regeneration after peripheral nerve injury is regulated by a variety of cells and cytokines.Previous studies mainly focused on how to promote peripheral nerve regeneration,but few studies focused on the factors that might inhibit nerve regeneration.Perineural and intraneural scarring at the repair site are important factors affecting axon regeneration,which reduce the growth rate of axons and limits the number of regrowing axons through the injury site.Through the in-depth study of nerve structure and regeneration mechanism,various methods were explored to inhibit the formation of scar tissue and promote peripheral nerve regeneration.Amnion is a biological tissue that is a semi-permeable membrane.Amnion is rich in collagen,laminin,proteoglycan and various of cell growth factors and other components.Amnion has anti-inflammatory and anti-fibrosis properties,and it could promote tissue regeneration.Modified surgical incision combined with amnion for carpal tunnel syndrome(CTS)could improve functional recovery of the affected limb and reduce the incidence of complications.In the clinical application process,amnion was easy to tear and had poor mechanical strength,poor extrusion resistance and rapid degradation rate.In order to overcome the deficiency of amnion,other materials should be introduced to improve the characteristics of amnion.Polycaprolactone(PCL)is a non-toxic biodegradable material with good compatibility.PCL has been used in skin reconstruction,bone tissue engineering,drug stents,tendon adhesion prevention and peripheral nerve regeneration.Therefore,our team prepared an amnion /PCL nanofibrous composite membrane to wrap the repair site of rat sciatic nerve,to evaluate the effect of the amnion/PCL nanofibrous composite membrane on inhibiting scarring proliferation at the repair site and promoting nerve regeneration.It is showed the grip and pinch strength of the affected limb were significantly enhanced,and symptom severity score of Boston carpal tunnel questionnaire(BCTQ-SSS)and of functional status score of Boston carpal tunnel questionnaire(BCTQ-FSS)scores were obviously improved,when modified incision combined with amnion were used for the treatment of CTS.This procedure improved functional recovery of the affected limb and reduced the incidence rate of wound pain and pillar pain.After the proximal and distal sciatic nerve stumps were reconnected using 10–0 Prolene epineurial sutures in a rat sciatic nerve transection model,the repaired site was wrapped with the amnion/PCL nanofibrous composite membrane.The amnion/PCL nanofibrous composite membrane alleviated the infiltration of macrophages and induced the activation of macrophages to M2 macrophages at the nerve repaired site.In addition,the amnion/PCL nanofibrous composite membrane decreased the expression of pro-inflammatory cytokines interleukin(IL)-6 and tumor necrosis factor-α(TNF-α)and increased the expression of anti-inflammatory cytokines IL-10 and IL-13 at the nerve repaired site.In addition,Amnion/PCL nanofibrous composite membrane slowly released cytokines,such as transforming growth factor-β(TGF-β),basic fibroblast growth factor(b FGF),platelet-derived growth factor(PDGF)and nerve growth factor(NGF)cytokines,which continuously diffused to the nerve repaired site through the pore structure of PCL nanofiber membrane and promoted the proliferation and migration of Schwann cells.Amnion/PCL nanofibrous composite membrane increased the number of axons passing through the nerve repaired site and promoted myelination of axons.PCL nanofiber showed good histocompatibility and promoted the proliferation and directional migration of Schwann cells,which promoted the myelination of axons.The amnion/PCL nanofibrous composite membrane improved the mechanical strength of amnion,reduced the degradation rate of amnion,and retained the bioactive molecules of amnion.The amnion/PCL nanofibrous composite membrane was used to wrap the nerve repaired site and to construct a neural regeneration chamber,which provided the microenvironment for peripheral nerve regeneration.The amnion/PCL nanofibrous composite membrane could inhibited scarring proliferation at the nerve repaired site and promoted the peripheral nerve regeneration and functional recovery of the affected limb.This study provides a new therapeutic method for promoting nerve regeneration,which provides a reliable theoretical basis and good technical support for clinical application.Part One Clinical effect of modified incision combined with amnion in the treatment of moderate to severe carpal tunnel syndromeObjective: To prevent the surgical complications of classic open carpal tunnel release and promote the functional recovery of nerve injury,a modified incision was performed to cut the transverse carpal ligament and release of the median nerve.The freeze-dried amnion graft was used to wrap the median nerve.The clinical efficacy of this surgical method for moderate to severe CTS was evaluated.Methods: A total of 64 patients with carpal tunnel syndrome were recruited from the Third Hospital of Hebei Medical University.According to the operation method,the patients were divided into modified incision combined with amnion group(group A,n=32)and traditional incision group(group B,n=32).The function of the affected limb was assessed before surgery and 3,6 months after surgery.The clinical efficacy of modified incision combined with amnion for moderate to severe CTS was evaluated by BCTQ-SSS and BCTQ-FSS,surgical incision length,surgical duration,grip strength,pinch strength,and the incidence rate of wound pain and pillar pain.Results: At 3 and 6 months after surgery,BCTQ-SSS scores of group A was lower than that of group B(P<0.01,P<0.01).At 3 and 6 months after surgery,BCTQ-FSS scores of group A was lower than group B(P<0.01,P<0.01).BCTQ-SSS scores and BCTQ-FSS scores showed a decreasing trend over time in both group A and group B,and the decreasing trend was more obvious in group A.The operation time of group A was significantly shorter than that of group B(P<0.01).The length of surgical incision in group A was significantly shorter than that in group B(P < 0.01)).The time of returning to work in group A was shorter than that in group B(P<0.01).At 6 months after surgery,the grip strength of group A was higher than that of group B(P <0.01).The pinch strength of group A was higher than that of group B(P<0.01).There were no wound pain and pillar pain in group A,however,there were 2 cases(6.3%)of wound pain and 3 cases(9.4%)of pillar pain in group B.Conclusion: The modified incision combined with amnion in the treatment of moderate to severe CTS has the advantages of short operation time and small incision,which significantly improved grip strength and pinch strength,reduced incidence of wound pain and pillar pain,reduced BCTQ-SSS and BCTQ-FSS scores and promoted the functional recovery of the affected limb.Part Two The effect of amnion/PCL nanofibrous composite membrane on macrophage polarization and scar formation in peripheral nerve regenerationObjective: Perineural and intraneural scarring at the nerve repaired site is an important factor inhibiting nerve regeneration,which not only limits the growth rate of regenerated axons,but also limits the number of regenerated axons.Macrophages also play an important role in the process of peripheral nerve regeneration.In this study,amnion/PCL nanofiber composite membrane was used to wrap the nerve repaired site in a rat sciatic nerve transection model,and its effect on scar formation and macrophage polarization of the nerve repaired site was determined.Methods: Fresh amnion was treated by freezing and vacuum drying to prepare freeze-dried amnion.PCL nanofibers were ejected on the two surfaces of amnion by electrostatic spinning technology to prepare amnion/PCL nanofibrous composite membrane.A total of 40 adult male SD rats were randomly divided into two groups: amnion/PCL nanofibrous composite membrane group(n=20)and control group(n=20).The sciatic nerve was transversely cut 15 mm distally from the sciatic notch.In the control group,the proximal and distal nerve stumps were reconnected using 10–0 Prolene epineurial sutures.In the amnion/PCL nanofibrous composite membrane group,the repaired site was wrapped with the amnion/PCL nanofibrous composite membrane after the epineurial sutures.General observation and histological observation were performed at 4 weeks after surgery.The relative expression levels of cytokines(IL-6,TNF-α,IL-10 and IL-13)in the nerve repaired site were analyzed by Quantitative RT-PCR.Results: At 4 weeks postoperative,loose adhesions were found around the sciatic nerve in the amnion/PCL nanofibrous composite membrane group.The surface of the nanofibrous membrane remained smooth.The Petersen’s adhesion grading scores of the amnion/PCL nanofibrous composite membrane group were less than those of the control group(P < 0.05).The number of intraneural macrophages in the amnion/PCL nanofibrous composite membrane group was significantly less than in the control group(P < 0.05).The number of M1 macrophages was significantly greater than M2 macrophages in the control group(P < 0.05).However,the number of M2 macrophages was significantly greater than M1 macrophages in the amnion/PCL nanofibrous composite membrane group(P < 0.05).The mean percentage of intraneural collagen staining in the amnion/PCL nanofibrous composite membrane group was significantly less than in the control group(P < 0.05).The expression of the pro-inflammatory cytokine IL-6 and TNF-α significantly decreased at the repair site in the amnion/PCL nanofibrous composite membrane group(P <0.05 for each cytokine).Moreover,the expression of the anti-inflammatory cytokines IL-10 and IL-13 significantly increased at the repair site in the amnion/PCL nanofibrous composite membrane group(P < 0.05 for each cytokine).Conclusion: Amnion/PCL nanofibrous composite membrane overcome the shortcomings of amnion(brittleness,low mechanical strength and fast degradation rate),and preserved the advantages of amnion.Amnion/PCL nanofibrous composite membrane was used to wrap the nerve repaired site and inhibited the scar formation in the nerve repaired site.Amnion/PCL nanofibrous composite membrane promoted macrophages polarizing to an M2 phenotype,which inhibited the expression of inflammatory cytokines and enhanced the expression of anti-inflammatory cytokines.Amnion/PCL nanofibrous composite membrane could promote the nerve regeneration and the function recovery of the effected limb.Part Three Experimental study of amnion/PCL nanofibrous composite membrane promoting peripheral nerve regenerationObjective: After peripheral nerve injury,the mechanism of nerve regeneration is complex,and the rate of nerve regeneration remains slow.Long-term denervation causes atrophy of the corresponding skeletal muscle.In this study,amnion/PCL nanofibrous composite membrane was used to wrap the nerve repaired site,and the effect of amnion/PCL nanofibrous composite membrane on nerve regeneration was analyzed.Methods: A total of 48 adult male SD rats were randomly divided into two groups: amnion/PCL nanofibrous composite membrane(n=24)and control group(n=24).The sciatic nerve was transversely cut 15 mm distally from the sciatic notch.In the control group,the proximal and distal nerve stumps were reconnected using 10–0 Prolene epineurial sutures.In the amnion/PCL nanofibrous composite membrane group,the repaired site was wrapped with the amnion/PCL nanofibrous composite membrane after the epineurial sutures.The outcomes of nerve regeneration in two groups were assessed by histological evaluation of sciatic nerve and gastrocnemius muscle,sciatic nerve index,electrophysiology examination and wet weight ratio of gastrocnemius muscles at 4,8,12 and 16 weeks.Results: At 8,12,and 16 weeks postoperative,the SFI of the amnion/PCL nanofibrous composite membrane group was significantly greater than that in the control group(P<0.05 for each time point).At 4 weeks postoperative,the density of myelinated axons in the amnion/PCL nanofibrous composite membrane group was significantly greater than that in the control group(P < 0.05).Furthermore,the average fiber diameter was significantly greater in the amnion/PCL nanofibrous composite membrane group in comparison with that in the control group(P < 0.05).The average myelin sheath thickness was significantly greater in the amnion/PCL nanofibrous composite membrane group than that in the control group(P < 0.05).At 12 and 16 weeks after never repair,the CMAP amplitude in the amnion/PCL nanofibrous composite membrane group was always significantly greater than that in the control group(P < 0.05,P < 0.05).At 12 weeks postoperative,DML in the amnion/PCL nanofibrous composite membrane group was significantly less than that in the control group(P < 0.05).Although no significance difference at 16 weeks(P > 0.05),DML in the amnion/PCL nanofibrous composite membrane group was less than that in the control group.At 12 weeks postoperative,the wet weight recovery ratio of gastrocnemius muscle in the amnion/PCL nanofibrous composite membrane group was higher than that in the control group(P < 0.05).The CSA of the gastrocnemius skeletal muscle was greater in the amnion/PCL nanofibrous composite membrane group(P < 0.05).At 16 weeks postoperative,the density of myelinated axons was significantly greater in the amnion/PCL nanofibrous composite membrane group than that in the control group(P < 0.05).Furthermore,the average diameter and myelin sheath thickness of the myelinated nerve fibers were significantly greater in the amnion/PCL nanofibrous composite membrane group than in the control group.Conclusion: Amnion/PCL nanofibrous composite membrane could promote the proliferation and migration of Schwann cells and axon regeneration,which improved the growth rate of axons and increased the number of regrowing axons.Amnion/PCL nanofibrous composite membrane could promote the recovery of nerve function.This study provided a new therapeutic method to promote nerve regeneration and provided theoretical basis and technical support for clinical treatment of nerve injury. |
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