WO2022110676A1 - 皮肤前体细胞诱导的施万细胞来源的囊泡在构建组织工程神经移植物中的应用 - Google Patents

皮肤前体细胞诱导的施万细胞来源的囊泡在构建组织工程神经移植物中的应用 Download PDF

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WO2022110676A1
WO2022110676A1 PCT/CN2021/093218 CN2021093218W WO2022110676A1 WO 2022110676 A1 WO2022110676 A1 WO 2022110676A1 CN 2021093218 W CN2021093218 W CN 2021093218W WO 2022110676 A1 WO2022110676 A1 WO 2022110676A1
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tissue
evs
scs
skp
nerve
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French (fr)
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贺倩茹
丁斐
施海燕
从猛
张琦
沈宓
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南通大学
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/383—Nerve cells, e.g. dendritic cells, Schwann cells
    • A—HUMAN NECESSITIES
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A61L27/3633—Extracellular matrix [ECM]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3675—Nerve tissue, e.g. brain, spinal cord, nerves, dura mater
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3878—Nerve tissue, brain, spinal cord, nerves, dura mater
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • A—HUMAN NECESSITIES
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/602—Type of release, e.g. controlled, sustained, slow
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction

Definitions

  • the invention belongs to the professional field of biomedicine, in particular to the application of vesicles (EVs) derived from Schwann cells (SKP-SCs) induced by skin precursor cells in the construction of tissue engineering nerve grafts.
  • EVs vesicles
  • SKP-SCs Schwann cells
  • peripheral nerve repair is performed by tension-free direct suture of nerve stumps, autologous or allogeneic nerve grafts, and tissue-engineered nerve grafts. Since the direct suture of the broken end is only suitable for local trauma with few defects, there are many disadvantages in the actual operation of autologous or allogeneic nerve transplantation. Therefore, the main research direction of peripheral nerve repair is to explore the construction of suitable tissue-engineered nerves. Two key elements of tissue-engineered nerves include biomaterial-made nerve conduits/scaffolds and implanted seed cells.
  • SCs Schwann cells
  • the most ideal seed cells for repairing peripheral nerve defects are Schwann cells (SCs).
  • SCs are myelinating cells of the peripheral nervous system and play a very important role in axon regeneration after nerve injury. It synthesizes and secretes a variety of neurotrophic factors, cell adhesion molecules and extracellular matrix to promote the survival of neuronal cell bodies, supports and guides axon regeneration, and finally wraps axons to form myelin sheaths to restore normal nerve function.
  • clinical sources of autologous SCs are limited, and obtaining SCs from autologous nerves will cause new damage to patients. Therefore, the search for seed cells to replace SCs is a research hotspot in the field of peripheral nerve regeneration.
  • Skin precursor cells are derived from neural crest stem cells in the embryonic stage and exist in the adult stage. They can be obtained from adult skin. They are rich in sources, convenient to obtain, and easy to culture and expand in vitro. Both rodent and human skin-derived precursor cells can differentiate into skin-derived precursor-induced SCs (SKP-SCs), which can be massively expanded in vitro and can promote nerve regeneration, making them ideal for tissue-engineered nerve grafts. seed cells.
  • SSP-SCs skin-derived precursor-induced SCs
  • seed cells seed cells.
  • the transplanted cells are mostly allogeneic sources, which will lead to immunogenicity after transplantation, which is not conducive to clinical promotion.
  • Cell-derived vesicles contain a variety of biologically active substances (protein, mRNA, microRNA, lnc RNA, and DNA, etc.), which are taken up by target cells after being released from cells to trigger corresponding biological effects.
  • a variety of cell-derived vesicles are an important way of intercellular interactions in the nervous system, and play an important role in the development and regeneration of peripheral nerves by regulating the regenerative microenvironment.
  • Vesicles can be obtained in large quantities in vitro, are easier to quality control, and have advantages in terms of safety and regulation. Compared to cells, it is immunologically inert and does not require autologous sources.
  • the vesicles can also be modified by in vitro culture to adjust the composition of the released components, which is more convenient for later quantitative use.
  • the use of vesicles to replace cells in regenerative medicine has been on the rise in recent years. Studies have shown that SCs-derived EVs play an important role in the repair process of peripheral nerve injury. However, the limited source of SCs restricts the further development of tissue-engineered nerve grafts.
  • the present invention provides a tissue engineered nerve graft for repairing peripheral nerve injury capable of promoting nerve axon regeneration and its application.
  • the invention adopts the method of tissue engineering, utilizes temperature-sensitive matrigel combined with SKP-SCs-derived EVs to construct a tissue-engineered nerve graft to repair peripheral nerve defects.
  • the invention not only overcomes the shortcoming of secondary injury caused by autologous nerve transplantation or acquisition of SCs, but also avoids the immunogenicity of allogeneic cell transplantation.
  • the bioactive factors that promote nerve regeneration released by EVs can be used to establish micro-organisms conducive to axon regeneration. In order to achieve the ideal goal of rapid nerve regeneration and functional recovery, it provides a feasible solution for clinical treatment.
  • the purpose of the present invention is to provide the application of Schwann cell-derived vesicles induced by skin precursor cells in the construction of tissue-engineered nerve grafts.
  • Another object of the present invention is to provide a tissue-engineered nerve graft, comprising a tissue-engineered nerve scaffold, Schwann cell-derived vesicles induced by skin precursor cells, and thermosensitive Matrigel.
  • the skin precursor cell-induced Schwann cell-derived vesicles are mixed with thermosensitive Matrigel, and filled in the inside and/or the pores of the tissue engineered neural scaffold in the form of gel.
  • the temperature-sensitive matrix glue of the present invention is liquid at 4° C., rapidly gels at 22-37° C., and its state change is reversible with temperature.
  • the concentration of the Schwann cell-derived vesicles induced by the skin precursor cells mixed with Matrigel is 5 ⁇ 10 8 particles/ ⁇ l.
  • the temperature-sensitive matrigel of the present invention is Matrigel matrigel (company: Corning, model: 354234).
  • Matrigel matrigel is liquid at 4°C, which is favorable for mixing with SKP-SCs-EVs, and becomes gelatinous at 37°C, preventing EVs from leaking out of the catheter.
  • the tissue engineered nerve scaffold of the present invention is a nerve conduit.
  • the tissue engineered nerve scaffold is made of biodegradable materials, preferably one or more of chitosan, silk fibroin, collagen, polylactic acid or polyglycolic acid.
  • the tissue-engineered nerve scaffold is a nerve conduit, preferably a chitosan conduit with a porous surface, such as a nerve conduit with a porous structure of 50-90%, a pore size of 50-300 ⁇ m, and a high tensile strength, a tubular body,
  • the inner diameter is 0.5-8mm, and the wall thickness is 0.1-3mm.
  • nanofiber silk fibroin catheter specifically can refer to the patent application number CN200910034583.9, publication number CN101664346, named as "the artificial nerve graft prepared by electrospinning and its preparation method and special device" described in method produced.
  • tissue engineered nerve graft of the present invention can be prepared by the following methods:
  • Skin precursor cell-induced Schwann cell-derived EVs are mixed with thermosensitive Matrigel (preferably in a 1:1 volume ratio) at 0-4°C, and the final concentration of SKP-SCs-EVs in the mixture is 5 ⁇ 10 particles/ ⁇ l, inject the mixture into the nerve conduit, then place the conduit at 22-37° C. (preferably 37° C.) for 30 minutes, and take out the mixture after it solidifies into a gel.
  • the preferred technical solution for the construction of the SKP-SCs-EVs tissue engineered nerve of the present invention is as follows: the degradable nerve conduit is sterilized and placed in a sterile petri dish, and trimmed to a desired length according to experimental requirements.
  • SKP-SCs-EVs were diluted with PBS and mixed with Matrigel at a volume ratio of 1:1.
  • the final concentration of SKP-SCs-EVs in the mixture was 5 ⁇ 10 8 particles/ ⁇ l. All operations were strictly performed on ice.
  • the well-mixed mixture of SKP-SCs-EVs and Matrigel was injected into the nerve conduit, and the conduit was placed at 37°C for 30 minutes and then taken out. The mixture solidified into a gel, which could be used to repair peripheral nerve defects.
  • the skin precursor cells are cultured to induce differentiation into SKP-SC, and the SKP-SC-derived EVs are isolated and collected after a large number of in vitro expansion and identified, and the collected SKP-SCs-EVs are marked with PKH-67 and then 4 ⁇ 10 8
  • the concentration of Particles/ml was incubated with sensory and motor neurons for 4h, and immunocytochemical staining showed that SKP-SCs-EVs could be internalized by sensory and motor neurons into the cell body.
  • SKP-SCs-EVs When SKP-SCs-EVs were added to DRG explants, sensory neurons or motor neuron medium at a certain concentration, SKP-SCs-EVs could significantly promote the growth of sensory and motor neuron neurites compared with the normal control group. Both SCs-EVs and SKP-SCs-EVs were added to the motor neuron medium at a concentration of 4 ⁇ 10 8 Particles/ml for 24 hours. Compared with the SCs-EVs group, SKP-SCs-EVs could significantly promote motor neurons. procession growth.
  • SKP-SCs-EVs were diluted with PBS and mixed with Matrigel in a volume ratio of 1:1, and the final concentration of SKP-SCs-EVs in the mixture was 5 ⁇ 10 8 particles/ ⁇ l, collect the upper buffer of the gel mixture on 1, 4, 7, 21, 28, 35, 42, 49 and 56d, respectively, measure the concentration of EVs in the upper release solution at each time point, and draw the release curve,
  • Matrigel can mediate the sustained release of EVs derived from SKP-SCs and provide continuous and stable nutritional support for the growth of neurites during regeneration.
  • the invention establishes a rat sciatic nerve 10mm defect model and randomly divides it into 6 groups: sham operation group, autologous nerve bridging group, silicone catheter group, SKP-SCs-EVs silicone tube group, chitosan catheter group, SKP-SCs-EVs shell Glycan catheter group. Footprint test was performed to analyze the sciatic nerve function index and thermal withdrawal test to detect the recovery of sciatic nerve function at 4 weeks after operation, respectively.
  • the present invention uses SKP-SCs-EVs combined with Matrigel to form a gel mixture to construct a tissue engineering nerve graft, which can not only prevent the direct use of EVs from causing poor repairing effect of fluid leakage, and Matrigel can also mediate SKP-SCs-EVs
  • the sustained release in vitro provides continuous and stable nutritional support for the growth of neurites during the regeneration process, and promotes the speed of nerve regeneration and the good recovery of function.
  • FIG. 1 Identification of SKP and differentiated SKP-SC cultured by light microscopy and immunocytochemical staining.
  • FIG. 1 Identification of SKP-SC-derived EVs.
  • FIG. 1 Identification of sensory and motor neurons by immunocytochemical staining.
  • SKP-SCs-EVs promote DRG and sensory neuron neurite outgrowth in vitro.
  • SKP-SCs-EVs were added to DRG explant culture medium for 1d, 3d and 5d (A) or SKP-SCs-EVs were added to sensory neuron culture medium for 12h (B), ⁇ -Tubulin III immunocytochemical staining was used to label neurites;
  • SKP-SCs-EVs promote motor neuron neurite outgrowth in vitro.
  • Fig. 8 Cumulative release (A) and release percentage (B) of EVs as a function of time in the mixture of SKP-SCs-EVs and Matrigel.
  • FIG. 9 Statistical chart of sciatic nerve function index (SFI) and thermal drawdown at 4w after operation.
  • A Statistical graph of sciatic nerve function index;
  • B Statistical graph of thermal drawdown.
  • the dorsal skin of neonatal SD rats was isolated about 1cm ⁇ 2cm, placed in dissection solution, the subcutaneous tissue was removed on ice, washed twice with dissection solution, chopped and transferred to a 15ml centrifuge tube, and 2ml of collagenase type XI (1mg/ml) and DNA were added. Enzyme, incubate in a 37°C 5% CO2 incubator; pipetting every 15min until the tissue digestion is cloudy, and adding 10ml of basal medium to terminate the digestion.
  • SKPs were cultured in serum-free medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), and the cells grew spherically.
  • bFGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • SKPs were digested with 2ml of collagenase type XI (0.5mg/ml) to form single cells and then adherent cultured. Serum medium induced SKPs to differentiate into SCs, and the changes in cell morphology during the induction were observed. Colonies were picked and expanded for culture. Immunocytofluorescence staining showed that SCs surface markers S100 ⁇ , GFAP, and P75 NTR were expressed, as shown in Figure 1B, D.
  • SKP-SCs-EVs were identified by transmission electron microscopy to have a typical cup-mouth morphology. Nano-tracer analysis (NTA) was used to detect the particle motion, concentration, particle size and distribution of EVs.
  • SKP-SCs-EVs were labeled with PKH-67 and incubated with sensory and motor neurons at a concentration of 4 ⁇ 10 8 Particles/ml for 4 h.
  • the neurons were labeled with ⁇ -Tubulin III to observe the internalization of EVs.
  • Immunocytochemical staining results It was found that SKP-SCs-EVs could be internalized into cell bodies by sensory and motor neurons, as shown in Figure 3.
  • DRG explant culture and sensory neuron culture DRG explants: 17d-18d pregnant SD rats, take out the fetuses and place them on ice, open the spinal canal under a dissecting microscope, remove the Spinal cord, L4-6DRG was carefully removed, placed in a 24-well plate pre-coated with polylysine, and cultured by adding Neurobasal complete medium (97% Neurobasal+2% B27+1% GluMAX). Cultivation of sensory neurons: the same as the DRG explant culture method, remove the adventitia as much as possible under the dissecting microscope of the DRG, and trim the nerve roots.
  • DRG explants 17d-18d pregnant SD rats, take out the fetuses and place them on ice, open the spinal canal under a dissecting microscope, remove the Spinal cord, L4-6DRG was carefully removed, placed in a 24-well plate pre-coated with polylysine, and cultured by adding Neurobasal complete medium (97% Neurobasal+2% B27+1% GluMAX
  • SKP-SCs-EVs were labeled with PKH-67 and added to DRG explants at a concentration of 2 ⁇ 10 8 Particles/ml. After culturing until the fifth day, it was found that the growth of neuronal processes in DRG explants could be significantly promoted. Compared with the obvious increase, as shown in Figure 5A,C. SKP-SCs-EVs were labeled with PKH-67 and then added to the sensory neuron culture medium at a concentration of 2 ⁇ 10 8 Particles/ml for 12 hours. The results showed that SKP-SCs-EVs could significantly promote the growth of sensory neuron processes, as shown in Figure 5B , shown in D.
  • the cell pellet was blown off with L15 and filtered through a 400-mesh sieve.
  • the filtered cells were slowly added to 5 ml of gradient centrifugation solution to make layers, and centrifuged at 2000 rpm for 20 min.
  • 5 mL of 9% Optiprep gradient separation solution was added and centrifuged at 2000 rpm for 20 min.
  • the liquid in the centrifuge tube was divided into three layers, and the middle layer contained spinal motor neurons. Carefully aspirate the middle layer, add 3 mL of medium and centrifuge at 1000 r/min for 5 min, discard the supernatant to remove cell debris.
  • SKP-SCs-EVs were added to motor neuron culture medium at a concentration of 4 ⁇ 10 8 Particles/ml and cultured for 12h, 24h and 36h, respectively. It was found that SKP-SCs-EVs could significantly promote the growth of motor neuron neurites, as shown in Figure 6. Show.
  • the SCs-EVs and SKP-SCs-EVs were added to the motor neuron medium at a concentration of 4 ⁇ 10 8 Particles/ml for 24 hours, respectively.
  • the results showed that the effect of SKP-SCs-EVs in promoting the growth of motor neuron neurites was significantly better than that of SCs.
  • -EVs as shown in Figure 7, SKP-SCs-EVs promoted motor neuron neurite outgrowth about 1.5 times longer than SCs-EVs.
  • SKP-SCs-derived EVs tissue-engineered nerves First, the degradable nerve conduits were sterilized and placed in a sterile petri dish, and trimmed to the desired length according to the experimental requirements.
  • the SKP-SCs-EVs obtained in Example 1 were diluted with PBS and mixed with Matrigel at a volume ratio of 1:1 (20 ⁇ l each), and the final concentration of SKP-SCs-EVs in the gel mixture was 5 ⁇ 10 8 particles / ⁇ l, all manipulations were performed on ice.
  • the homogeneous mixture of SKP-SCs-EVs and Matrigel was injected into the nerve conduit, and the conduit was placed at 37°C for 30 min. The mixture was solidified into a gel, which can be used to repair peripheral nerve defects.
  • SKP-SCs-derived EVs tissue-engineered nerves The SKP-SCs-EVs obtained in Example 1 were diluted with PBS and mixed with Matrigel at a volume ratio of 1:1 (20 ⁇ l each), and the mixture was The final concentration of SKP-SCs-EVs was 5 ⁇ 10 8 particles/ ⁇ l.
  • the upper layer buffer of the gel mixture was collected on 1, 4, 7, 21, 28, 35, 42, 49 and 56 days, respectively, and the concentration of EVs in the upper release solution at each time point was measured by NTA, and the cumulative number and percentage of EVs released particles were calculated. , draw the corresponding release curve, the results suggest that Matrigel can mediate the sustained release of SKP-SCs-derived EVs, providing continuous and stable nutritional support for the growth of neurites during regeneration, as shown in Figure 8.
  • a rat model of 10 mm sciatic nerve defect was established and randomly divided into 6 groups: sham operation group, autologous nerve bridging group, silicone catheter group, SKP-SCs-EVs silicone catheter group, chitosan catheter group, SKP-SCs-EVs chitosan group catheter set. Footprint test was performed to analyze the sciatic nerve function index and thermal withdrawal test to detect the recovery of sciatic nerve function at 4 weeks after operation, respectively.
  • the sciatic nerve function index results showed that the sciatic nerve function index of the SKP-SCs-EVs tissue engineered nerve (including silicone catheter and chitosan catheter) group was significantly better than that of the silicone catheter group and the chitosan catheter group alone, as shown in Figure 9A.

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Abstract

皮肤前体细胞诱导的施万细胞来源的囊泡在构建组织工程神经移植物中的应用。采用组织工程的方法，利用Matrigel基质胶结合SKP-SCs来源-EVs构建了组织工程神经移植物用于修复周围神经缺损，既克服了自体神经移植或获取施万细胞造成二次伤害的缺点，又避免同种异体细胞移植的免疫原性，通过EVs释放的促进神经再生的生物活性因子建立有利于轴突再生的微环境，以达到神经快速再生并功能恢复的理想目标。

Description

皮肤前体细胞诱导的施万细胞来源的囊泡在构建组织工程神经移植物中的应用 技术领域
本发明属于生物医学专业领域，具体涉及皮肤前体细胞诱导的施万细胞(SKP-SCs)来源的囊泡(EVs)在构建组织工程神经移植物中的应用。
背景技术
周围神经损伤的修复及功能重建一直是临床治疗的一个难题。临床上采用神经断端无张力直接缝合、自体或异体神经移植、以及组织工程化神经移植物等方式进行神经修复。由于断端直接缝合只适用于缺损少的局部创伤，自体或异体神经移植实际操作中有诸多弊病。因此，周围神经修复的主要研究方向是探索构建合适的组织工程化神经。组织工程神经的两大关键要素包括生物材料制备的神经导管/支架和植入的种子细胞。
修复周围神经缺损最理想的种子细胞为施万细胞(Schwann cells，SCs)，SCs是周围神经系统的成髓鞘细胞，在神经损伤后的轴突再生过程中起着非常重要的作用。其合成和分泌多种神经营养因子、细胞粘附分子和细胞外基质等来促进神经元胞体存活，支持和引导轴突再生并最终包裹轴突形成髓鞘，使神经恢复正常功能。但临床上自体SCs来源有限，并且从自体神经获取SCs会给患者造成新的损伤。故而寻找替代SCs的种子细胞，是周围神经再生领域的研究热点。
皮肤前体细胞(SKPs)来自胚胎期神经嵴干细胞，并且存在于成体期，可从成人皮肤中获得，来源丰富，取材方便，在体外易于培养和扩增。啮齿动物和人的皮肤来源前体细胞均可分化为皮肤来源前体诱导的SCs(SKP-SCs)，可在体外进行大量扩增，并可促进神经的再生，是组织工程神经移植物中理想的种子细胞。但是细胞移植进入体内后其归属、生物学效应和安全性等问题目前并不完全清楚，并且移植细胞多为同种异体来源，移植后会导致免疫原性不利于临床推广。而细胞来源的囊泡(EVs)，包含多种生物活性物质(蛋白质、mRNA、microRNA、lnc RNA和DNA等)，从细胞释放后被目标细胞摄取从而引发相应的生物学效应。近年的研究表明多种细胞来源的囊泡是神经系统细胞间相互作用的重要方式，通过调节再生微环境在周围神经发育和再生过程中起着重要作用。囊泡可在体外大量获得，更易于进行质量控制，在安全性和监管方面更具有优势。与细胞 相比，其又具有免疫学惰性，不需要自体来源。囊泡还可结合体外培养修饰以调节释放成分的组成，更利于后期的量化使用。近年来在再生医学中使用囊泡来代替细胞的疗法的正在兴起。研究表明SCs来源的EVs在周围神经损伤后修复过程中发挥着重要作用，然而SCs由于来源受限，制约了其进一步用于组织工程神经移植物的发展。
发明内容
本发明针对现有技术不足提供了一种能够促进神经轴突再生的用于修复周围神经损伤的组织工程神经移植物及其应用。本发明采用组织工程的方法，利用温敏性基质胶结合SKP-SCs来源EVs构建组织工程神经移植物以修复周围神经缺损。本发明既克服了自体神经移植或者获取SCs造成二次伤害的缺点，又能够避免同种异体细胞移植的免疫原性，通过EVs释放的促进神经再生的生物活性因子建立有利于轴突再生的微环境，以达到神经快速再生并功能恢复的理想目标，为临床治疗提供可行方案。
本发明目的在于提供皮肤前体细胞诱导的施万细胞来源的囊泡在构建组织工程神经移植物中的应用。
本发明另一目的在于提供一种组织工程神经移植物，包括组织工程神经支架、皮肤前体细胞诱导的施万细胞来源的囊泡和温敏性基质胶。优选的，所述皮肤前体细胞诱导的施万细胞来源的囊泡和温敏性基质胶混合，以凝胶的形式填充于组织工程神经支架内部和/或孔隙中。本发明所述的温敏性基质胶在4℃为液态，22-37℃温度环境下快速成胶，其状态变化随温度可逆。
优选的，所述皮肤前体细胞诱导的施万细胞来源的囊泡与Matrigel基质胶混合后的浓度为5×10 8particles/μl。
优选的，本发明所述的温敏性基质胶为Matrigel基质胶(公司：Corning，型号:354234)。Matrigel基质胶在4℃时为液体，有利于和SKP-SCs-EVs混合，置于37℃即变成凝胶状，防止EVs漏出导管。
本发明所述组织工程神经支架为神经导管。所述组织工程神经支架由生物可降解材料制成，优选壳聚糖、丝素蛋白、胶原、聚乳酸或聚羟基乙酸中的一种或几种。
一个优选的方案，所述组织工程神经支架为神经导管，优选表面多孔的壳聚糖导管，如50-90％、孔径50-300μm的具有多孔结构、抗拉强度高的神经导管， 管状本体，内径为0.5-8mm，壁厚0.1-3mm。具体可参照专利申请号为CN201110324474.8，名为“组织工程神经移植物及其用途”中所述的方法制得。或者优选纳米纤维蚕丝丝素蛋白导管，具体可参照专利申请号为CN200910034583.9，公开号为CN101664346，名为“静电纺丝制备的人工神经移植物及其制备方法和专用装置”中所述的方法制得。
本发明所述的组织工程神经移植物可采用如下方法制备而成：
在0-4℃将皮肤前体细胞诱导的施万细胞来源的EVs与温敏性基质胶混合(优选以1:1体积比混合)，混合物中SKP-SCs-EVs终浓度为5×10 8particles/μl，将混合物注入神经导管中，再将导管置于22-37℃(优选37℃)静置30min，待混合物凝固成凝胶状后取出，即得。
本发明SKP-SCs-EVs组织工程神经的构建优选的技术方案如下：将可降解神经导管灭菌处理后置于无菌培养皿中，根据实验需求修剪成所需长度。
SKP-SCs-EVs使用PBS稀释后与Matrigel基质胶两者以1:1体积比混合，混合物中SKP-SCs-EVs终浓度为5×10 8particles/μl，所有操作均严格在冰上进行。将混合均匀的SKP-SCs-EVs和Matrigel基质胶混合物注入神经导管中，将导管置于37℃静置30min后取出，混合物凝固成凝胶状，可用于修复周围神经缺损。
本发明培养皮肤前体细胞诱导分化为SKP-SC，体外大量扩增后分离并收集SKP-SC来源EVs并进行鉴定，将收集的SKP-SCs-EVs采用PKH-67标记后以4×10 8Particles/ml浓度与感觉和运动神经元共孵育4h，免疫细胞化学染色结果发现SKP-SCs-EVs可被感觉与运动神经元内化进入胞体。将SKP-SCs-EVs以一定浓度加入DRG植块、感觉神经元或运动神经元培养基中，与正常对照组相比，SKP-SCs-EVs可以显著促进感觉和运动神经元突起的生长。将SCs-EVs和SKP-SCs-EVs分别均以4×10 8Particles/ml浓度加入运动神经元培养基中培养24h，与SCs-EVs组相比，SKP-SCs-EVs可以显著促进运动神经元突起生长。
本发明SKP-SCs来源EVs组织工程神经中EVs释放检测：SKP-SCs-EVs使用PBS稀释后与Matrigel基质胶两者以1:1体积比混合，混合物中SKP-SCs-EVs终浓度为5×10 8particles/μl，分别于1、4、7、21、28、35、42、49和56d收集凝胶混合物上层缓冲液，测定每个时间点上层释放液中EVs的浓度，绘制释放曲线，结果提示Matrigel基质胶可介导SKP-SCs来源EVs缓释，为再生过程中神经突起的生长提供持续稳定的营养支持。
本发明建立大鼠坐骨神经10mm缺损模型并随机分为6组：假手术组、自体神经桥接组、硅胶导管组、SKP-SCs-EVs硅胶管组，壳聚糖导管组，SKP-SCs-EVs壳聚糖导管组。分别与术后4周进行足迹试验分析坐骨神经功能指数和热敏回撤实验检测坐骨神经功能恢复情况。结果显示SKP-SCs-EVs组织工程神经(包括硅胶导管和壳聚糖导管)组坐骨神经功能指数明显优于单独硅胶导管组和壳聚糖导管组，并且热敏回撤时间明显缩短，与阳性对照自体神经桥接组相比无明显差异。提示SKP-SCs-EVs组织工程神经内的EVs能在体内缓释营养因子，促进周围神经再生与功能恢复。
本发明优点：
本发明使用SKP-SCs-EVs结合Matrigel基质胶形成凝胶混合物构建组织工程神经移植物，既可以防止直接使用EVs导致液体外漏修复效果不佳，Matrigel基质胶又可介导SKP-SCs-EVs的体外缓释，为再生过程中神经突起的生长提供持续稳定的营养支持，促进神经再生的速度和功能的良好恢复。
附图说明
图1SKP和诱导分化的SKP-SC培养光镜和免疫细胞化学染色鉴定图。A：SKP培养至第三代，细胞呈球形集落生长；B：SKP免疫细胞化学染色鉴定图；C：SKP-SC培养光镜图；D：SKP-SC免疫细胞化学染色鉴定图。
图2SKP-SC来源EVs鉴定图。A：SKP-SC来源EVs电镜图；B：NTA检测EVs粒径；C：Western Blot检测EVs标记物CD9、CD63、CD81和TSG101。
图3PKH-67标记EVs后检测感觉与运动神经元对其的内化作用。
图4感觉与运动神经元免疫细胞化学染色鉴定图。A：β-Tubulin III标记感觉神经元；B：β-Tubulin III和ChAT标记运动神经元。
图5SKP-SCs-EVs体外促进DRG和感觉神经元突起生长。DRG植块培养液中加入SKP-SCs-EVs培养1d、3d和5d(A)或感觉神经元培养液加入SKP-SCs-EVs12h(B)，β-Tubulin III免疫细胞化学染色标记神经突起；DRG轴突面积(C)和最长突起长度(D)统计图，*p<0.05，**p<0.01，***p<0.001vs对照组。
图6SKP-SCs-EVs体外促进运动神经元突起生长。A：运动神经元培养液加入SKP-SCs-EVs12h、24h和36h，β-Tubulin III免疫细胞化学染色标记神经元突起；B：平均突起长度统计图，*p<0.05，**p<0.01vs对照组。
图7 SCs-EVs和SKP-SCs-EVs体外促进运动神经元突起生长。运动神经元培养液加入SCs-EVs(A)和SKP-SCs-EVs(B)培养24h，β-Tubulin III免疫细胞化学染色标记神经元突起；C：平均突起长度统计图，**p<0.01vs SCs-EVs。
图8 SKP-SCs-EVs与Matrigel基质胶混合物随时间变化EVs累计释放量(A)和释放百分比(B)。
图9术后4w坐骨神经功能指数(SFI)和热敏回撤统计图。A：坐骨神经功能指数统计图；B：热敏回撤统计图。
具体实施方式
以下通过实施例说明本发明的具体步骤，但不受实施例的限制。
在本发明中所使用的术语，除非另有说明，一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例并参照数据进一步详细描述本发明。应理解，这些实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
在以下实施例中，未详尽描述的各种过程和方法是本领域中公知的常规方法。
下面结合具体实施例对本发明进一步说明。
实施例1 SKP-SCs的培养和EVs的收集
1.SKP-SCs的培养和分化
分离新生SD大鼠背部皮肤约1cm×2cm，置于解剖液中，于冰上剔除皮下组织，解剖液漂洗2遍后剪碎移入15ml离心管，加入2mlⅪ型胶原酶(1mg/ml)及DNA酶，37℃5％CO2培养箱孵育；每隔15min吹打一次，直至组织消化呈云雾状，加入10ml基础培养基终止消化。4℃1200rpm离心5min，弃上清，加入10ml培养基重悬组织，400目筛网过滤，4℃1200rpm离心7min后弃上清，1ml培养基重悬细胞；细胞计数，以5.0×10 4个/ml，接种于大皿中。应用添加碱性成纤维生长因子(bFGF)和表皮生长因子(EGF)的无血清培养液培养SKPs，细胞呈球形生长，传至二代后进行鉴定，如图1A，C所示。SKPs用2ml Ⅺ型胶原酶(0.5mg/ml)消化为单细胞后贴壁培养，应用含腺苷酸环化酶激活剂(Forskolin)、外源性生长因子(Heregulin-1β，HRG)的无血清培养基诱导SKPs向SCs分化，观察诱导过程中细胞形态的变化，挑取集落并扩大培养，免疫细胞荧光染色发现表达SCs表面标志S100β、GFAP、P75 NTR，如图1B，D 所示。
2.SKP-SCs来源EVs的分离与鉴定
将SKP-SCs接种于10cm培养皿中，待细胞密度长至80-90％左右，换无血清培养基继续培养48h收集上清，4℃，500g离心5min除去细胞碎片后收集上清，过0.22μm滤器。采用exoEasy Maxi Kit(QIAGEN)进行细胞外囊泡的提取。如图2A所示，SKP-SCs-EVs经透射电镜鉴定具有典型的杯口状形态。采用纳米示踪分析(NTA)检测EVs的颗粒运动、浓度、粒径大小和分布，结果发现SKP-SCs-EVs浓度达到4.9×10 10Particles/ml，粒径大小集中在80-220nm范围内，如图2B所示。Western Blot结果显示EVs表达其表面标志物CD9、CD63、CD81和TSG101，如图2C所示。
将收集的SKP-SCs-EVs采用PKH-67标记后以4×10 8Particles/ml浓度与感觉和运动神经元共孵育4h，β-Tubulin III标记神经元观察EVs内化，免疫细胞化学染色结果发现SKP-SCs-EVs可被感觉与运动神经元内化进入胞体，如图3所示。
实施例2考察SKP-SCs-EVs对感觉与运动神经元突起生长的促进作用
1.背根神经节(DRG)植块培养和感觉神经元的培养：DRG植块：将孕17d-18d的SD大鼠，取出胎鼠置于冰上，解剖镜下打开椎管，移除脊髓，小心取出L4-6DRG，置于预先包被多聚赖氨酸的24孔板中，加入Neurobasal完全培养基(97％Neurobasal+2％B27+1％GluMAX)培养。感觉神经元的培养：同DRG植块培养方法，取出DRG解剖镜下尽量去除外膜，修剪神经根。将移入0.25％胰酶和0.5g/L胶原酶中，37℃消化30min。加入DMEM+10％FBS终止消化，1 000r/min离心5min，弃上清。加入Neurobasal完全培养基(97％Neurobasal+2％B27+1％GluMAX)重悬细胞。将细胞悬液按1×10 5/mL种植到预先包被多聚赖氨酸的24孔板中。培养的第3天，加入2 0mg/L 5-氟尿嘧啶抑制增殖类非神经元细胞，维持48h后更换成Neurobasal完全培养基，之后每3d半量换液，β-Tubulin III标记感觉神经元纯度鉴定见图4A。
SKP-SCs-EVs采用PKH-67标记后以2×10 8Particles/ml浓度加入DRG植块中，培养至第5天发现可以显著促进DRG植块中神经元突起的生长，突起面积与对照组相比明显增大，如图5A，C所示。SKP-SCs-EVs采用PKH-67标记后以2×10 8Particles/ml浓度加入感觉神经元培养液中培养12h，结果发现 SKP-SCs-EVs可以显著促进感觉神经元突起的生长，如图5B，D所示。
2.运动神经元的培养：将孕13.5d的SD大鼠颈椎脱臼处死，取出胎鼠，于解剖镜下打开椎管，去除背根神经节和被膜，分离出胎鼠腹侧脊髓，置于盛有冰冷解剖液的培养皿中。用显微剪剪成0.5cm 3大小的组织块后转移至15mL离心管中，加入1ml 0.125％胰酶将组织吹散，37℃消30min，加入DMEM+10％FBS终止消化，1 000r/min离心5min，弃上清。细胞沉淀用L15吹散，400目筛网过滤。将过滤后的细胞缓慢加入5ml梯度离心液中使其分层，2000rpm离心20min。加入5mL 9％Optiprep梯度分离液，2000rpm离心20min，离心管中的液体分为3层，其中中间层含有脊髓运动神经元。小心将中间层吸出，加入3mL培养基1000r/min离心5min，弃上清以去除细胞碎片。加入DMEM+10％FBS重悬后接种细胞，4h后观察细胞贴壁情况以及活力，换预热的神经元培养基(97％Neurobasal+2％B27+1％GluMAX)，之后每3d半量换液，β-Tubulin III和乙酰胆碱转移酶(ChAT)标记运动神经元纯度鉴定见图4B。
SKP-SCs-EVs以4×10 8Particles/ml浓度加入运动神经元培养液中分别培养12h，24h和36h，结果发现SKP-SCs-EVs可以显著促进运动神经元突起的生长，如图6所示。
将SCs-EVs和SKP-SCs-EVs分别以4×10 8Particles/ml浓度加入运动神经元培养基中培养24h，结果显示，SKP-SCs-EVs促进运动神经元突起生长的效果显著优于SCs-EVs，如图7所示，SKP-SCs-EVs促进运动神经元突起生长的长度是SCs-EVs的约1.5倍。
实施例3本发明所述SKP-SCs来源EVs组织工程神经的构建
1.构建SKP-SCs来源EVs组织工程神经：首先将可降解神经导管灭菌处理后置于无菌培养皿中，根据实验需求修剪成所需长度。实施例1中获得的SKP-SCs-EVs使用PBS稀释后与Matrigel基质胶两者以1:1体积比(各20μl)混合，凝胶混合物中SKP-SCs-EVs终浓度为5×10 8particles/μl，所有操作均在冰上进行。将混合均匀的SKP-SCs-EVs和Matrigel基质胶混合物打入神经导管中，将导管置于37℃，静置30min后取出，混合物凝固成凝胶状，可用于修复周围神经缺损。
2.SKP-SCs来源EVs组织工程神经中EVs释放检测：实施例1中获得的SKP-SCs-EVs使用PBS稀释后与Matrigel基质胶两者以1:1体积比(各20μl) 混合，混合物中SKP-SCs-EVs终浓度为5×10 8particles/μl。分别于1、4、7、21、28、35、42、49和56d收集凝胶混合物上层缓冲液，采用NTA测定每个时间点上层释放液中EVs的浓度，计算累积EVs释放颗粒数及百分比，绘制相应释放曲线，结果提示Matrigel基质胶可介导SKP-SCs来源EVs缓释，为再生过程中神经突起的生长提供持续稳定的营养支持，如图8所示。
实施例4本发明所述SKP-SCs来源EVs组织工程神经在修复神经缺损中的应用
建立大鼠坐骨神经10mm缺损模型并随机分为6组：假手术组、自体神经桥接组、硅胶导管组、SKP-SCs-EVs硅胶管组，壳聚糖导管组，SKP-SCs-EVs壳聚糖导管组。分别与术后4周进行足迹试验分析坐骨神经功能指数和热敏回撤实验检测坐骨神经功能恢复情况。坐骨神经功能指数结果显示SKP-SCs-EVs组织工程神经(包括硅胶导管和壳聚糖导管)组坐骨神经功能指数明显优于单独硅胶导管组和壳聚糖导管组，如图9A所示。热敏回撤实验结果显示术后4周，SKP-SCs-EVs组织工程神经(包括硅胶导管和壳聚糖导管)组与硅胶导管组和壳聚糖导管组相比，回撤时间明显缩短，与阳性对照自体神经桥接组相比无明显差异，如图9B所示。结果提示SKP-SCs-EVs组织工程神经内的EVs能在体内缓释促进周围神经再生与功能恢复。

Claims (10)

  1. 皮肤前体细胞诱导的施万细胞来源的囊泡在构建组织工程神经移植物中的应用。
  2. 一种组织工程神经移植物，其特征在于包括组织工程神经支架、皮肤前体细胞诱导的施万细胞来源的囊泡和温敏性基质胶。
  3. 如权利要求2所述的组织工程神经移植物，其特征在于所述皮肤前体细胞诱导的施万细胞来源的囊泡和温敏性基质胶混合，以凝胶的形式填充于组织工程神经支架内部和/或孔隙中。
  4. 如权利要求3所述的组织工程神经移植物，其特征在于所述凝胶中皮肤前体细胞诱导的施万细胞来源的囊泡的浓度为5×10 8particles/μl。
  5. 如权利要求2-4任一项所述的组织工程神经移植物，其特征在于所述温敏性基质胶为Matrigel基质胶。
  6. 如权利要求2所述的组织工程神经移植物，其特征在于所述组织工程神经支架为神经导管。
  7. 如权利要求2所述的组织工程神经移植物，其特征在于所述组织工程神经支架由生物可降解材料制成。
  8. 如权利要求7所述的组织工程神经移植物，其特征在于所述生物可降解材料选自壳聚糖、丝素蛋白、胶原、聚乳酸或聚羟基乙酸中的一种或几种。
  9. 如权利要求8所述的组织工程神经移植物，其特征在于所述组织工程神经支架为表面多孔的壳聚糖导管，所述导管孔隙率为50-90％、孔径50-300μm，内径为0.5-8mm，壁厚0.1-3mm。
  10. 如权利要求2-9任一项所述的组织工程神经移植物的制备方法，其特征在于在0-4℃将皮肤前体细胞诱导的施万细胞来源的囊泡与温敏性基质胶混合制备成浓度为5×10 8particles/μl的混合物，注入神经导管中，再将导管置于22-37℃，待混合物凝固成凝胶状后，制备完成。
PCT/CN2021/093218 2020-11-25 2021-05-12 皮肤前体细胞诱导的施万细胞来源的囊泡在构建组织工程神经移植物中的应用 WO2022110676A1 (zh)

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