| In clinical practice,repair of large segmental defects of peripheral nerves and bone has been challenging.When the tissue defect is too large to be repaired,it is often necessary to repair the defect by autograft.However,problems such as donor shortage and donor area complications have limited its application.Tissue scaffolds of different materials have the role of supporting tissue structure and guiding tissue growth,and are an important alternative to autograft in clinical practice.The defects of single material tissue scaffolds have only tissue supporting and guiding effects and lack induction effects.Therefore,in order to improve the pro-repair ability of scaffolds,functionalized scaffolds were created.Functionalized scaffolds are structurally bionic,can load stem cells or induction factors,and can better promote tissue repair.Studies have shown that electrical stimulation can promote peripheral nerve and bone regeneration.Therefore,conferring electrical activity/conductivity to tissue scaffolds can improve their ability to promote tissue repair.The currently applied epidermal electrical stimulation is inefficient,and transcutaneous electrical stimulation has a short operating time window and risk of infection.The research of non-invasive electrical stimulation provides a new technical basis to solve the above problems,but it is limited by the non-degradable scaffold material or uncontrollable electrical stimulation time/intensity.Is it possible to select a biological material with excellent performance and design a novel electrical stimulation method to give precise,continuous and controllable electrical stimulation to tissues,thus promoting tissue regeneration?Silk protein material has good biocompatibility,degradability,mechanical strength and processability.It is a good material for building multifunctional tissue scaffolds.If a silk protein tissue scaffold can be combined with a wireless translation device with better performance to give more precise and controlled electrical stimulation to tissues,it will provide a better option for treating large segments of peripheral nerve and bone tissue defects.Therefore,the purposes of this study are to produce a degradable tissue scaffold based on silk protein that can deliver stable,precise,and wireless electrical stimulation via inductive coupling,and to test its efficacy in promoting nerve and bone defect repair.The study is divided into three parts.Part Ⅰ: Fabrication and performance testing of composite electrode silk protein scaffoldOBJECTIVE: To develop composite electrode silk protein scaffolds,including self-coiling nerve conduits and bone repair membranes,and to optimize and test their physicochemical and biological properties to provide a research basis for experiments on nerve tissue and bone tissue repair.METHODS: Optimization of material preparation:(1)The silk protein solution is extracted and the silk protein membrane is prepared by the currently accepted extraction process,and the molecular weight of the protein is controlled by adjusting the degumming time,which in turn adjusts the mechanical properties of the membrane;(2)Mechanical tests,including Young’s modulus and fracture stress,were performed on the protein films.The protein membrane with the best mechanical properties was screened for subsequent experiments.(3)Fixing the crystalline orientation in the stretching direction by directional pre-stretching to produce super-shrinkable protein films that shrink when exposed to water and stable protein films that do not deform;(4)Overlaying super shrink film with ordinary protein film to produce a double-sided,self-curling film.Select the production parameters for the best curl effect by observing its curl performance;(5)Vaporized metal electrodes on the surface of silk film by Shadow mask process,mainly including induction coils,connecting wires and fork-like electrodes,among which some fork-like electrodes were vaporized onto the self-curling film,and the best image vaporization was achieved by adjusting the line width and line spacing;(6)Assembled composite electrode supports by anisotropic conductive adhesive film(ACF)welding and pressing process.Physicochemical properties characterization:(1)Morphological characterization: the protein membrane structure and electrode morphology were characterized by surface scanning electron microscopy(SEM);(2)Mechanical Properties: mechanical tester to characterize the mechanical properties of the material: stress-strain test curve,fracture stress and Young’s modulus;(3)Degradation properties: the silk protein film was immersed in 0.1 mg/ml protease K solution to observe its degradation properties;(4)Electrical properties: including circuit impedance changes,the effectiveness and stability of electrical signal wireless transmission.Biosafety characterization:(1)Cellular safety of the material was detected by CCK-8 and live-dead cell staining;(2)Tissue safety was evaluated by HE staining of each organ after scaffold implantation into animals.RESULTS: Optimization of material preparation:(1)Different types of silk protein films were prepared,and after characterization of mechanical properties,the Young’s modulus of the protein film with a degumming time of 30 min was 9.63 MPa,and the fracture stress was 5.25 MPa,which was higher than other protein films;(2)Stable film and self-curling film were prepared,and the self-curling film was successfully self-curled into tubular structure after soaking in water for 10min;(3)When the coil electrode specification is coil diameter 1cm,line width 200μm,line distance 300μm,the evaporation pattern is clear and the stability is high;(4)When the dissolution parameter is90℃ for 3s and the solidification parameter is 120℃ for 13 s,the connection between each component after ACF welding and pressing is firm and the well-connected.Physicochemical properties characterization:(1)Mechanical properties characterization suggested that Young’s modulus and fracture stress of the stable film were7.4 MPa and 6.0 MPa,respectively,and Young’s modulus and maximum stress of the self-curled film were 22.8 MPa and 7.6 MPa,respectively;(2)Silk protein film showed complete degradation after 9 days of immersion in 0.1% protease K solution;(3)Image evaporation is clear and accurate when the coil diameter is 1cm,the line width is 200μm,and the line distance is 300μm;(4)The initial resistance of the electrode was 26 Ω,which could be maintained for at least 1 month without elevation in the body fluid environment.The wireless transmission effect of inductively coupled electrical stimulation is stable.The bidirectional square wave(20Hz,10V)at the transmitter can output bidirectional pulse stimulus(20Hz,0.56V)at the induction terminal.Moreover,the intensity of the electrical signal at the transmitting end is linearly correlated with the output stimulus intensity at the corresponding end(slope = 0.612).Biosafety characterization: The activity of PC12 cells and BMSCs cultured in material extract was not significantly different from that in ordinary medium.The staining of living/dead cells on the surface of the material also did not show significant cell death.No significant pathological changes were found in each organ section after 12 weeks implantation of composite electrode silk protein.CONCLUSION: In this part of the study,silk protein tissue scaffolds with electrodes were successfully developed through screening and optimization of fabrication parameters.The scaffolds have good mechanical properties and degradation properties,can effectively and stably transmit electrical stimulation,have good biocompatibility,and are qualified for further pro-tissue repair experiments.Part Ⅱ: Study on the effectiveness of composite electrode silk protein catheter to promote peripheral nerve repairOBJECTIVES:(1)To examine the effect and potential mechanism of composite electrode silk protein conduit in promoting axon growth through cytological experiments;(2)To explore the efficacy of scaffold in promoting the repair of 10 mm nerve defect through animal experiments.METHODS: Cytology experiments: The experiment was divided into three groups:Control group,Silk group and Silk+ES material group.(1)primary dorsal root ganglion(DRG)cells and tissue explants were extracted,and cells/explants in the Silk+ES group were given electrical stimulation(200m V/mm,20 Hz,pulsed,1h/d,5d)by wireless transmission;(2)Cells were identified by immunofluorescence staining of β3-tubulin and cell adhesion and extension were evaluated by SEM,and the effects of electrical stimulation on axon growth were explored;(3)β3-tubulin specifically labels nerve axons,and mitotracker specifically labels mitochondria.The effects of electrical stimulation on mitochondrial density and mitochondrial transport capacity in DRG cells were observed under confocal microscopy.Animal experiments: Ten millimeters SD rat sciatic nerve defect model was established,and the experimental animals were randomly divided into 4groups: Defect group,Autograft group,Conduit group and Conduit+ES group.Nerve electrical stimulation(200m V/mm,20 Hz,pulsed,1h/d,14d)was given to the Conduit+ES group by wireless transmission.(1)Dynamic and static footprints of rats were collected at4,8 and 12 weeks postoperatively to calculate dynamic sciatic function index(SFI)and static sciatic nerve index(SSI);(2)Nerve electrical signals were collected and analyzed by BL-420 N biosignal acquisition and analysis system;(3)Sciatic nerve was collected 12 weeks after operation,and the nerve repair effect was evaluated by nerve HE staining,immunofluorescence staining and transmission electron microscopy(TEM);(4)Both gastrocnemius muscles were collected 12 weeks after surgery,and the degree of target muscle atrophy was evaluated by muscle wet weight,HE staining and Masson staining.RESULTS: Cytological experiments:(1)Cells of each group adhered and extended well,while the length of nerve axons of DRG cells and explants in the Silk+ES group was significantly higher than that of other groups(P<0.05).The number of new nerve axons in tissue blocks also increased significantly(P<0.05),and the degree of complexity was higher;(2)Axonal mitochondrial density of DRG cells of Silk+group was significantly higher than that of the control group(P<0.05),showing a significant increase in the number of motile mitochondria.Animal experiments:(1)Both SFI and SSI in Conduit+ES group were higher than those in Conduit+ES group(P<0.05),and there was no statistical difference between Conduit+ES group and Autograft group at 12W;(2)Neuroelectrophysiological results showed that the amplitude of composite action potential in Conduit+ES group was higher than that in Conduit group(P<0.05),the latency was lower than that in Conduit group(P<0.05),and the nerve conduction velocity was faster than that in Conduit group(P<0.05);(3)Morphological evaluation showed that there were more new nerves in Conduit+ES group than in Conduit group,which was close to the autograft group,and the axons arrangement is more orderly and more convergent;(4)The degree of atrophy and fibrosis of the target muscles in the Conduit+ES group was less than that of the Conduit group(P<0.05)and higher than that of the autograft group(P<0.05).CONCLUSION: The composite electrode silk protein nerve conduit can significantly promote the structural repair and functional recovery of peripheral nerves through the wireless transmission of electrical stimulation.At the same time,it can accelerate the re-neuralization of target muscle and slow down the atrophy and fibrosis of target muscle.The repair effect is close to that of autograft.This accelerative effect may be related to the new conduit’s ability to enhance the transport capacity of axon mitochondria through wireless electrical stimulation.Part Ⅲ: Study on the effectiveness of composite electrode silk protein bone repair membrane to promote bone repairOBJECTIVES:(1)The effect of composite electrode silk protein membrane on promoting osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)was verified by cytological experiments.(2)The effectiveness of composite electrode bone repair membrane in promoting skull defect repair was verified by animal experiments.METHODS: Cytological experiments: The experiments were divided into 3 groups:Control group,Silk group and Silk+ES group.(1)BMSCs were extracted from SD rats,and the cells of the Silk+ES group were electrically stimulated by wireless transmission(200m V/cm or 200 m V/mm,20 Hz,pulsed,1h/d,7d);(2)The expression of osteogenic differentiation indicators was detected by reverse transcription-polymerase chain reaction(q RT-PCR);(2)The degree of ossification and mineralization of BMSCs was evaluated by alkaline phosphatase staining(ALP)and alizarin red staining.Animal experiments:5-mm cranial defect model was established in SD rats,and the experimental animals were randomly divided into 3 groups: Defect group,Membrane group and Membrane+ES group.Nerve electrical stimulation(200m V/cm,20 Hz,pulsed,1h/d,14d)was given to the Membrane+ES group by wireless transmission.(1)The animals were sacrificed at 8 weeks after surgery,and the bone defect repair was evaluated by micro-CT,HE staining and Masson staining;(2)The expression of bone repair-related indicators was evaluated by immunohistochemistry and immunofluorescence staining.RESULTS: Cytological experiments:(1)q RT-PCR results showed that the expression of osteogenic differentiation-related indicators was significantly up-regulated in the BMSCs of the Silk+ES group(P<0.05);(2)ALP straining results showed that the ALP activity of BMSCs in the Silk+ES group was significantly higher than that in other groups;(3)Alizarin red staining showed that calcium deposition of BMSC in Silk+ES group was significantly higher than that in other groups(P<0.05).Animal experiments:(1)Morphological evaluation showed that the new bone of Membrane+ES group was significantly higher than that of Membrane group and Defect group(P<0.05),and the bone density,number and thickness of trabecular bone were also significantly higher(P<0.05),and the bone are more mature;(2)The results of immunofluorescence staining showed that the expression of bone repair factors in Membrane+ES group was significantly higher than that in Membrane group and Defect group.CONCLUSION: By adjusting the electrical stimulation parameters,the composite electrode silk protein bone repair membrane was able to promote osteogenic differentiation and mineralization of BMSCs through wireless transmission of electrical stimulation,which could effectively promote the repair of cranial defects in rats in vivo.This indicates that the tissue scaffold developed in this study can promote the repair of different tissues through.SummaryIn this study,a controllable-adjustable-degradable functional tissue scaffold for multi-tissue repair was constructed by composite surface electrode based on silk protein material.The new tissue scaffold has several advantages as follows:(1)Material: tissue scaffolds based on silk protein have good physical and chemical properties,biological safety and plasticity,and can be made into a variety of structures to adapt to different tissues,such as self-curling nerve conduit and bone repair membrane;(2)Electrical stimulation delivery: after compounding surface electrodes,the tissue scaffold can transmit electrical stimulation wirelessly and stably through inductive coupling,and the intensity and frequency of stimulation can be controlled,so that the electrical stimulation parameters can be adjusted for different tissues.(3)Promoting multi-tissue repair: both in vivo and in vitro experiments confirmed that the tissue scaffold can effectively promote the regeneration and repair of large peripheral nerve and bone tissue defects by adjusting stimulation parameters.ConclusionThe silk functionalized tissue scaffold developed in this study has good physicochemical and biological properties.The scaffold was able to give stable and controlled electrical stimulation to the tissues,which effectively promoted the repair of peripheral nerve and bone tissues.This provides a new idea for the clinical treatment of large segments of peripheral nerve and bone tissue defects.In addition to this,the plasticity of the scaffold and the controllability of electrical stimulation make it potential to be used in the clinical treatment of multi-tissue defect repair. |
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