Hydrogel Biomaterials For Nerve Injury Repair

Background:Repair and regeneration of long-segment nerve injury has been the research hotspot and difficulty in the field of nerve regeneration for many years.When the patient's peripheral nerve injury has a large defect,and the tension of the direct nerve suture is too large,so that the joint can not be sutured excessively,nerve transfer and autologous nerve transplantation are mainly used to repair the continuity of the injured nerve.Regardless of the choice of the two surgical procedures,patients are faced with the problem of sacrificing the nerves of their own secondary donor sites.With the development of multidisciplinary integration,hydrogel biomaterials have attracted much attention in the field of medical research in the past decade.Hydrogel is a hydrophilic polymer material with a network structure.It has excellent biocompatibility and ion transport ability.Its three-dimensional cross-linked structure can absorb water and shape to obtain a correspondingly strong scaffold structure.It is widely used in the research of bionic materials and artificial tissues,and the repair of peripheral nerve damage is also an important application direction.This research focuses on the photosensitive hydrogel methacryloyl(gelatin methacryloyl,GelMA).Through two experimental systems,the application of this hydrogel biomaterial in the repair of peripheral nerve injury and brachial plexus nerve injury is explored.Objectives:The first experimental system uses 3D printed composite tissue engineering nerve conduits to promote peripheral nerve regeneration.Neural guidance conduit(NGC)is currently the most promising alternative to autologous nerve transplantation because it can seal the distal and proximal ends of the defective nerve and provide a suitable environment for axon regeneration.Using photosensitive hydrogel methacryloyl gelatin(gelatin methacryloyl,GelMA)and surface-modified magnesium microparticles,3D printing technology was used to prepare composite tissue engineering nerve conduits,and the physical and chemical properties were characterized and implanted in animals to verify the NGC promotion The effect of peripheral nerve regeneration.The second experimental system uses GelMA combined with surfacemodified magnesium microparticles and Schwann cells(schwann cells,SCs)composite tissue hydrogel to promote nerve regeneration after brachial plexus avulsion.After brachial plexus nerve root avulsion occurred,due to the massive loss of nerve tissue and the formation of glial scars,which hindered the extension and regeneration of nerve axons,nerve root replantation was used for treatment.The excellent biocompatibility of GelMA,combined with surface-modified magnesium particles and SCs for co-treatment,can observe the effect of promoting nerve regeneration after replantation of brachial plexus nerve root avulsion.Materials and Methods:In the first experimental system,NGC was first prepared,and magnesium particles(Mg)and sodium hydroxide solution(Na OH aq)were bathed in a 75°C water bath for 24 hours to form Mg-OH;Mg-OH and 3-aminopropyltrimethoxysilane((3-Aminopropyl)trimethoxysilane,APTMS)ethanol solution was mixed and shaken for 10 hours to form Mg-APTMS;Mg-APTMS and polyethylene glycol(PEG),1-(3-dimethylaminopropyl)-3-Ethylcarbodiimide hydrochloride(1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,EDC),Nhydroxysuccinimide(N-Hydroxysuccinimide,NHS)are mixed and shaken for 10 hours to form Mg-PEG;Mg-PEG and fibronectin(fibronectin,Fn)PBS solution is heated for 2 hours to form Mg-PEG-Fn;Mg-PEG-Fn is treated with EDC and NHS,and after reaction for 2 hours,Fn and heparin solution are added dropwise in equal volume,Mg-PEG-FH is formed and the final product is obtained.The products of each stage are characterized by scanning electron microscope(SEM)and Fourier transform attenuated total internal reflectance fourier transform infrared spectroscopy(ATRFTIR).Secondly,the characterized magnesium particles(Mg-PEG-FH)were added as the solute of liquid GelMA,and the composite tissue engineering nerve conduit was made by 3D printing technology,and it was characterized as having no obvious cytotoxicity.Finally,this NGC was used to treat a 10 mm length defect model of the rat sciatic nerve.The experimental animals were divided into 5 groups: Autograft group,GelMA group,GelMA+Mg-PEG-FH group,GelMA+SCs group,GelMA+MgPEG-FH+SCs group.The motor function,neuroelectrophysiology,and histology of regenerated sciatic nerve in rats were evaluated 3 months after operation.In the second experimental system,the characterized magnesium particles(Mg-PEG-FH)obtained in the first experimental system were added as the solute of liquid GelMA,and SC was added to make a composite tissue hydrogel.This kind of composite tissue hydrogel is used to treat brachial plexus nerve root avulsion and replantation model in rats.In the operation,the right cervical nerves 5 to 7 posterior roots,6 cervical nerves anterior roots are replanted,and the right neck avulsion 5-Neck 7 anterior roots.The experimental animals were divided into 5 groups: Autograft group,GelMA group,GelMA+Mg-PEG-FH group,GelMA+SCs group,GelMA+Mg-PEG-FH+SCs group.The Terzis grooming test(TGT)was performed on the motor function of the rat's forelimbs at 2 weeks after the operation,once a week.The rat biceps brachii muscle electrophysiological examination was performed 6 weeks after the operation.Hematoxylin-eosin staining(HE)staining was performed on the injured biceps of the rat.Results:The first experimental system successfully modified the surface of Mg particles.The NGC combined with GelMA and Mg-PEG-FH was prepared by 3D printing technology.In vitro experiments confirmed that it has the ability to guide the proliferation of Schwann cells.Monthly motor function evaluation results,rat sciatic functional index(sciatic functional index,SFI)Autograft group,GelMA group,GelMA+Mg-PEG-FH group,GelMA+SCs group,GelMA+Mg-PEG-FH+SCs group,respectively For,-35.71±2.678,-74.01±10.75,-70.78±3.71,-77.2±2.986,-45.84±3.222,Autograft group and GelMA+Mg-PEG-FH+SCs group were significantly higher than GelMA group,GelMA+Mg-PEG-FH group and GelMA+SCs group;rat compound muscle action potential(CMAP)amplitude Autograft group,GelMA group,GelMA+Mg-PEG-FH group,GelMA+SCs group,GelMA+ The average values of the Mg-PEG-FH+SCs group were 6.52 m V,3.66 m V,5.24 m V,4.56 m V,5.53 m V,GelMA+Mg-PEG-FH+ SCs group and Autograft group were significantly larger than the other three groups;Autograft group,GelMA The diameters of myelinated axons in the +MgPEG-FH+SCs group,GelMA+Mg-PEG-FH group,GelMA+SCs group and GelMA group were 4.50±1.00,4.22±0.53,1.60±0.55,2.89±0.52,1.78± 0.49?m.The autograft group and GelMA+Mg-PEG-FH+SCs group have no significant difference in the myelinated axon diameter of regenerated nerves.Both of them are significantly larger than the GelMA+Mg-PEG-FH group,GelMA+SCs group and GelMA group..The second experimental system successfully modeled the rat brachial plexus root avulsion replantation model,and the TGT scores of each group rose steadily after the operation.Autograft group,GelMA group,GelMA + Mg-PEG-FH group,GelMA + SCs group The mean TGT scores of the affected limb of the rats in the GelMA + Mg-PEG-FH + SCs group were 4.8,3.3,4.3,4.0,and 4.7 at the 6th week.The GelMA+Mg-PEG-FH+SCs group and Autograft There is no significant difference between the two groups,which are significantly higher than the GelMA+Mg-PEG-FH group,GelMA+SCs group and GelMA group;the electrophysiological results of rat biceps muscles show: Autograft group,GelMA group,GelMA+Mg-PEG-FH Group,GelMA+SCs group,GelMA+Mg-PEGFH+SCs group 6 weeks after the operation of the average action potential amplitude: 1.46,1.00,1.24,1.09 and 1.29mv;Autograft group,GelMA+Mg-PEG-Compared with the GelMA group,the amplitude of the FH+SCs group increased statistically(*P?0.05);the amplitude of the Autograft group increased significantly(**P?0.01);the results of HE staining of the injured biceps brachii muscle of the rat at 6 weeks postoperatively Display: Autograft group,GelMA group,GelMA+MgPEG-FH group,GelMA+SCs group,GelMA+Mg-PEG-FH+SCs group The average muscle diameters are: 43.45,28.64,37.60,34.14,40.08?m(Figure 3.4 AE).Among them: Although the diameter of the biceps brachii muscle fiber in the GelMA+SCs group was larger than that in the GelMA group,it was not statistically significant.The muscle fiber diameter of the GelMA+Mg-PEG-FH group,GelMA+Mg-PEG-FH+SCs group and Autograft group was significantly larger than that of the Autograft group,and it was statistically significant(***P?0.001,****P? 0.001).Conclusions:The first experimental system uses 3D printing technology to prepare NGC,which has better manufacturing freedom than traditional manufacturing and shaping technology,especially for complex structures,lowering the threshold of individual customization technology;successful application of photosensitive hydrogel GelMA and surface modification The composite tissue engineered NGC made of magnesium microparticles(Mg-PEG-FH)and filled with SCs can better promote the regeneration of the rat's sciatic nerve defect and promote the recovery of the motor function of the rat's affected side.In the second experimental system,the application of GelMA composite surface-modified magnesium particles and SCs hydrogel can promote nerve root replantation after brachial plexus avulsion in motor function,histology and electrophysiology.regeneration.In summary,the rational application and exploration of hydrogel biomaterials is expected to further broaden human cognition in the field of nerve regeneration and provide new possibilities for humans to repair extreme length nerve defects.