CN-122005920-A - Acellular tissue engineering nerve conduit carrying dental pulp stem cell exosome and preparation method and application thereof

Abstract

The invention provides a cell-free tissue engineering nerve conduit carrying dental pulp stem cell exosomes, and a preparation method and application thereof, relates to the technical fields of biomedical engineering and nerve regeneration medicine, and aims to solve the problems of poor mechanical property, low bionic degree, high degradation rate, insufficient bioactivity and the like of the conventional nerve conduit. The technical key point of the invention is that the invention provides a cell-free tissue engineering nerve conduit carrying dental pulp stem cell exosome, the nerve conduit is obtained by loading DPSC-Exo with enhanced function of overexpression let-7i-5p by a nanofiber conduit, the porous nanofiber conduit is a porous conduit obtained by passing a polyvinyl alcohol (PVA) -Chitosan (CS) -graphene (G) electrostatic spinning membrane through a coiled tube and crosslinking, and the nucleic acid sequence of let-7i-5p in the DPSC-Exo is shown as SEQ NO. 1.

Inventors

• SUN JINGXUAN
• HE LINA
• SUN XIANGYU
• NIU YUMEI
• PAN SHUANG
• ZHANG LIN

Assignees

• 哈尔滨医科大学

Dates

Publication Date

20260512

Application Date

20260228

Claims (10)

1. 1. The acellular tissue engineering nerve conduit carrying the dental pulp stem cell exosome is characterized in that the nerve conduit is obtained by loading DPSC-Exo with function enhanced by over-expressing let-7i-5p by a nanofiber conduit, the porous nanofiber conduit is a porous conduit obtained by winding and crosslinking a polyvinyl alcohol (PVA) -Chitosan (CS) -graphene (G) electrostatic spinning film, and the nucleic acid sequence of the let-7i-5p in the DPSC-Exo is shown as SEQ NO. 1.
2. 2. The nerve conduit according to claim 1, wherein the method of preparing the nerve conduit comprises the steps of: (1) Mixing PVA solution, CS solution and graphene dispersion liquid, uniformly stirring to obtain spinning liquid, and adding the spinning liquid into an electrostatic spinning machine to prepare the PVA/CS/G electrostatic spinning film; (2) Winding the PVA/CS/G electrostatic spinning film on a mould to form a tubular structure, and crosslinking in glutaraldehyde steam to obtain a porous nanofiber catheter; (3) Preparation and Loading of DPSC-Exo: a. let-7i-5p mimetic transfection by synthetic chemical modification of let-7i-5p mimic while preparing negative control mimic, transfecting mimic into DPSCs using liposomal transfection reagents or electrotransfection; b. The exosome is prepared by culturing DPSCs, collecting the conditioned medium, and obtaining DPSC-Exo suspension through centrifugation, separation and purification; c. Freeze-drying the DPSC-Exo suspension to obtain DPSC-Exo freeze-dried powder; d. Exosome loading, namely loading DPSC-Exo freeze-dried powder on the porous nanofiber catheter to obtain the DPSC-Exo loaded cell-free tissue engineering nerve catheter.
3. 3. The nerve conduit according to claim 2, wherein in step (1) the concentration of PVA solution is 8% -15%, the concentration of CS solution is 4%, the concentration of graphene dispersion is 0.05-0.10 mg/mL, and the volume ratio of PVA graphene to CS solution is 9:1.
4. 4. The nerve conduit of claim 3, wherein the DPSC-Exo loaded cell free tissue engineering nerve conduit has a porosity of 60% -80%, an average pore size of 0.5-2.0 μm, an inner diameter of 1-4 mm, and a wall thickness of 0.2-0.5 mm.
5. 5. The nerve conduit according to claim 4, wherein the loading of the DPSC-Exo lyophilized powder in the step (3) c is performed by preparing a DPSC-Exo lyophilized powder complex solution, and then fully adsorbing the DPSC-Exo on the surface and the inside of the pores of the porous nanofiber conduit by oscillation adsorption.
6. 6. The nerve conduit according to claim 4, wherein the step (3) c is characterized in that the DPSC-Exo freeze-dried powder is loaded on the porous nanofiber conduit, specifically, the DPSC-Exo freeze-dried powder is uniformly mixed with porous gelatin sponge powder or porous hydrogel precursor solution to prepare a mixed carrier, and then the mixed carrier is filled or coated in the inner cavity of the conduit, and the mixed carrier is solidified to form the active inner core for loading the exosomes.
7. 7. The use of DPSC-Exo according to claim 1 for the preparation of a biomaterial, wherein the biomaterial is a nerve conduit for promoting peripheral nerve regeneration after nerve injury.
8. 8. The nanofiber catheter of claim 1.
9. 9. Use of a nanofiber catheter as claimed in claim 8 for the preparation of a biomaterial for promoting peripheral nerve regeneration after nerve injury.
10. 10. The use according to claim 9, wherein the biological material is a nerve conduit.

Description

Acellular tissue engineering nerve conduit carrying dental pulp stem cell exosome and preparation method and application thereof Technical Field The invention relates to the technical fields of biomedical engineering and nerve regeneration medicine, in particular to a cell-free tissue engineering nerve conduit carrying dental pulp stem cell exosomes, and a preparation method and application thereof. Background Peripheral nerve injuries such as facial nerve are common in clinic, and have limited regeneration capacity, especially discrete injuries. The existing clinical gold standard 'autologous nerve transplantation' has inherent defects of damaged donor areas, limited sources and the like. Tissue engineering nerve conduits offer a promising clinical treatment for nerve regeneration. The ideal nerve conduit needs to have 1) good biocompatibility, degradability and proper degradation rate, 2) a microstructure of the bionic extracellular matrix to simulate a regeneration microenvironment, 3) sufficient mechanical support performance to better guide directional growth of the nerve axon, and 4) a bioactive component capable of loading and slowly releasing the nerve regeneration promotion. The existing catheter is mainly focused on material and structure optimization, but in terms of biological activity endowment, the traditional strategy (such as directly carrying living stem cells) has the problems of low cell survival rate, immune rejection, ethical safety, inconvenient storage and transportation and the like, and the clinical transformation of the catheter is limited. In recent years, paracrine effects of stem cells (e.g., dental pulp stem cells), particularly vesicles such as exosomes secreted by the stem cells, have gradually been demonstrated to play an important role and have significant application advantages. The dental pulp stem cells are derived from neural crest and are 'homologous' to neural tissue, and the exosomes naturally carry a large amount of active substances (neurotrophic factors, mirnas, etc.) which are beneficial to nerve regeneration. Can simultaneously act on a plurality of links of repair, such as inhibiting inflammation, protecting schwann cells, promoting angiogenesis and axon guidance, and can simulate the treatment effect of stem cells in a cell-free mode, promote nerve regeneration, and avoid the potential risk of living cell treatment. In terms of materials, the electrostatic spinning technology can prepare the nanofiber scaffold with the highly bionic natural extracellular matrix. The Chitosan (CS) has good biocompatibility and nerve repair promoting property, and the polyvinyl alcohol (PVA) has good spinnability, and the spinnability can be improved by blending the Chitosan (CS) and the PVA. The addition of graphene (G) can significantly enhance the mechanical properties and conductivity of the material, the latter being particularly advantageous for regeneration of electrically excitable tissues such as nerves. Therefore, a novel nerve conduit which combines a bionic nanofiber structure and excellent mechanical/conductive performance and carries an efficient and safe cell-free active ingredient of dental pulp stem cell exosome (DPSCs-Exo) is developed, has important significance for promoting the development of peripheral nerve regeneration treatment, and has not been reported at present. Disclosure of Invention The invention aims to solve the technical problems that: The existing nerve conduit has the problems of poor mechanical property, low bionic degree, high degradation rate, insufficient biological activity and the like. The invention adopts the technical scheme for solving the technical problems: Aiming at the problems of unstable bioactive components, immunogenicity risk or complex preparation and storage of the nerve conduit, the invention provides a novel tissue engineering nerve conduit which does not contain living cells, has high safety and stable activity and is convenient to store and transport. Specifically, the invention provides a composite catheter which takes PVA/CS/G nanofiber as a structural substrate and functional dental pulp stem cell exosome freeze-dried powder which is remarkably enriched with let-7i-5p as a core bioactive component, and a controllable and efficient preparation method thereof, which are used for safely and efficiently repairing peripheral nerve injuries such as facial nerves. The invention provides a cell-free tissue engineering nerve conduit carrying a functionalized dental pulp stem cell exosome, which is obtained by loading DPSC-Exo with a nanofiber conduit and over-expressing let-7i-5p, wherein the porous nanofiber conduit is a porous conduit obtained by carrying out coil tube and crosslinking on a polyvinyl alcohol (PVA) -Chitosan (CS) -graphene (G) electrostatic spinning film, and the nucleic acid sequence of the let-7i-5p in the DPSC-Exo is shown as SEQ NO. 1. Further, the preparation method of the nerve conduit comprises the