| Background:The maintenance of organ function and bodily processes is regulated not only by humoral factors,but also by neural signaling.Neural deficits are a common clinical problem worldwide,arising from various factors such as peripheral nerve injury,neural loss in engineered tissues,and surgical complications,leading to uncontrolled pathophysiological processes and resulting in sensory and/or motor disabilities as well as failure of engineered tissue/organ transplants.These conditions severely impact patients’quality of life and impose significant socio-economic burdens.However,with the emergence of neural tissue engineering and regenerative medicine,promising therapeutic approaches are now available for repairing and/or replacing damaged tissues.The nervous system operates through a sophisticated network of electrical signals to enable communication between neurons and other cell types.Neural tissue is composed of electrically active nerve cells that transmit electrochemical signals.In vertebrates,the peripheral nervous system（PNS）consists of bundles of nerves,comprising many axonal fibers that transmit input and output signals to and from the central nervous system（CNS）at speeds of approximately 100 meters/second.Thus,the nervous system represents a complex combination of electrical,ionic,chemical signals,and specialized cellular structures.Over the past decade,organic bioelectronics（OBE）has made significant progress in interacting with medical biology at multiple levels.The core of this field is the establishment of electrical connections with the nervous system to regulate and control the immune response,physiological state of organs,and systems.Organic bioelectronic materials,also known as conductive polymers（CPs）,areπ-conjugated organic semiconducting materials with a range of unique characteristics.Their dual conduction properties of electrons and ions are closely related to the transmission modes of biological systems.By establishing a close coupling through electron-ion charge compensation,they can reduce local electrochemical impedance between live cells and biomaterials.Therefore,CPs offer an ideal connection for the bio-region and have attracted considerable attention in biomedical applications.However,regulating the microstructure of the material at the subcellular level to provide a more seamless interface between the conductive substrate and cells remains a significant challenge.Therefore,investigating how to assemble CPs into interface-adaptive structures and provide electrical connections for neural interfaces is a key scientific issue in addressing signal connections in tissue engineering bio-regions.Objectives:Our research focuses on developing molecular assembly structures for neural cell interface connections and signal transmission.We aim to explore in vivo assembly synthesis of conductive polymers to enhance neural interface conductivity and promote neural regeneration and reconstruction.We design fixed-enzyme-triggered and constructed molecular assemblies to investigate their application in interface modification of tissue-engineered blood vessels to promote their neuralization.Additionally,we aim to study degradable conductive polymer materials synthesized in vivo using tissue metabolism mechanisms.Methods:1.In Vivo Assembly of Synthetic Conductive Polymers to Facilitate Nerve Tissue RepairThis section of the experiment involves using immunofluorescence to detect superoxide generation and related enzymes in tissues under conditions of tissue injury,in order to determine the necessary conditions for the oxidative polymerization of aniline.Cell viability of polymer monomers and injected gels is tested using AM/PI staining.An animal nerve injury model is established,and after injection of the gel precursor at different time points,the microstructure and morphology of the polymer after in situ polymerization are analyzed using scanning electron microscopy.Atomic force microscopy and Raman spectroscopy are used to determine the synthesis of the polymer in the tissue.The function and effect of in situ assembly of conductive microvesicles（CMVs）in vivo are evaluated,using animal behavioral tests to assess their effect on behavior,electrophysiological detection to determine their conduction at the neural interface,and immunofluorescence staining and TEM to evaluate their effect on tissue repair and neural regeneration.2.In Vitro Construction of Conductive Microvesicles and Modification of Tissue-Engineered Vascular InterfaceSubcellular-scale microvesicles were constructed by lipid extrusion and encapsulation of catalase enzyme（CAT）,and CMVs were constructed in vitro by interface polymerization.The tissue-engineered vascular scaffold was activated by EDC/NHS and the conductive polymer was modified onto the outer membrane of the scaffold to construct an interface capable of conducting and transmitting ion information.The neurotization of tissue-engineered vascular scaffold was achieved through primary neuron transplantation technique.The effect of interface construction on tissue-engineered vascular scaffolds was evaluated using rat carotid artery transplantation technique,vascular ultrasound,HE staining,and other methods.3.In vivo fate of conductive microvesicles and in vivo assembly of degradable polymers3.The in vivo fate and assembly of conductive microvesicles（CMVs）with degradable conductive polymersThe movement and localization of CMVs in vivo were observed using histological techniques such as hematoxylin and eosin staining and immunofluorescence,as well as confocal laser scanning microscopy of whole glass slides,with an aim to explore their localization pathways.Additionally,the synthesis and degradation mechanisms of degradable conductive polymers were studied in vitro,and their ability to construct in vivo was evaluated using an animal model,with the morphology of the resulting structures observed using scanning electron microscopy and confocal laser scanning microscopy.Furthermore,the functionality of the resulting structures was determined using electrophysiological testing.Results:1.Morphology and characterization of conductive polymers assembled in vivoIn vitro,aniline monomer can be polymerized under the action of H2O2 and CAT enzyme,and CAT enzyme can accelerate the synthesis of polyaniline.In vivo,a large amount of reactive oxygen species（ROS）will be produced after tissue damage,and the expression of CAT enzyme will also increase,which constitutes the conditions for the polymerization of aniline monomer in vivo.In order to reduce the biotoxicity of aniline monomer,ANI was fused with sodium alginate into an injectable sol by using hydrogen bond and electrostatic interaction force.Through the rat sciatic nerve crush model,Alg/ANI sol was injected,and it was found that a lot of granular substances were aggregated in the tissue space in vivo one day after the operation.Confirmed by CLSM and SEM,the particles are microvesicles with a size of about 2-3 microns.After atomic force microscopy and laser microscopy Raman showed that these microvesicle-like substances are in vivo assembled conductive polymer microvesicles（CMVs）2.In vitro construction of cellularized microvessels（CMVs）and their interface modification to promote nerve regeneration in TEBVsThrough decellularized vascular matrix materials,small-diameter tissue-engineered blood vessels（TEBVs）were obtained.By polymerizing PANI on the outer surface of TEBVs and transplanting DRG neurons hydrogel,the patency of TEBVs was improved.Moreover,neural fibers were observed to grow into the outer membrane of TEBVs in a short time.To simulate the in vivo assembly of CMVs,CAT enzyme was successfully encapsulated into liposomes（Lipo@CAT）using liposome extrusion technology and interface synthesis,and conductive polymers were assembled on its surface.CLSM observation showed that dark polyaniline and polypyrrole were polymerized on the surface of Lipo@CAT.Additionally,CMVs could be activated by EDC/NHS and modified on the outer surface of TEBVs.Animal vascular transplantation and ultrasound examination showed that the experimental rats with PPy A-CMVs modification had the best patency of transplanted blood vessels.3.The in vivo fate and assembly of conductive microvesicles（CMVs）with degradable conductive polymersThe long-term effects of neural tissue repair and material integration were investigated.Hematoxylin and eosin staining（HE）and confocal laser scanning microscopy（CLSM）revealed that non-degradable CMVs assembled in vivo migrated to the neural outer membrane,thanks to the active transport of capillaries and lymphatic vessels.Therefore,tunable degradable conductive polymers can be applied to in vivo assembled CMVs.By conducting in vitro and in vivo experiments on the polymerization and degradation of pyrrole and pyrrole carboxylic acid,it was found that PPy A can be depolymerized under the influence of p H=7.4 and H2O2.Additionally,PPy A can also be assembled into CMVs in vivo,providing more fiber connections between tissues,enhancing neural conduction velocity,and reducing local electrical impedance of the nerve.Conclusion:1.Under the action of CAT enzyme and local tissue ROS,aniline monomers can be assembled and polymerized in vivo.The conductive polymer assembled and synthesized in the body is a microvesicle with a diameter of about 2-3 microns,which is a subcellular-scale conductive microvesicle.In vivo assembly of conductive microvesicles can promote the recovery of rat mobility,form tight junctions between tissue cells,thereby increasing the conduction speed of nerve electrical signals,and is conducive to nerve regeneration and remodeling of the local tissue microenvironment.2.Conductive polymer,PPy A CMVs modified TEBV interface can promote the maintenance of vascular patency.3.Non-degradable PANI microvesicles were removed to the epineurium through capillary lymphatic vessels.The in vivo polymerized PPy A CMVs can be degraded in vivo for a long time. |
