CN113384752A

CN113384752A - Exosome-based nerve dissociation repair material and preparation method thereof

Info

Publication number: CN113384752A
Application number: CN202110652864.1A
Authority: CN
Inventors: 易西南; 曹旭东; 崔天威; 冯仁军; 樊好飞
Original assignee: Hainan Medical College
Current assignee: Hainan Medical College
Priority date: 2021-06-11
Filing date: 2021-06-11
Publication date: 2021-09-14

Abstract

The invention discloses a preparation method of a nerve dissociation repair material based on exosomes, which comprises the steps of taking a freeze-dried mixed biosynthesis cellulose membrane, soaking the freeze-dried mixed biosynthesis cellulose membrane in normal saline at 25 ℃ for 5min, dividing the cellulose membrane into three layers by using forceps, punching four holes at two ends of a single-layer BBC membrane, sterilizing the single-layer BBC membrane at high temperature and high pressure, airing the moisture on the BBC membrane, placing the single-layer BBC membrane in a sterile six-hole plate, and placing 4 mu L of sterile exosome solution (each mu L contains 1.9 multiplied by 10)⁹One exosome) is slowly and evenly drippedAnd (4) applying the membrane on a single layer of BBC membrane and drying the membrane in the air to obtain the exosomal embedded neurolysis repair material EXO-BBC membrane. When the EXO-BBC membrane prepared by the invention is applied to a rat sciatic nerve dissociation repair test, the effect of the EXO-BBC membrane is obviously superior to that of an intramuscular injection exosome + BBC membrane or the BBC membrane is used alone, and the EXO-BBC membrane is safe and practical. Therefore, the EXO-BBC membrane prepared by the invention has wide clinical application prospect in the field of nerve dissociation repair.

Description

Exosome-based nerve dissociation repair material and preparation method thereof

Technical Field

The invention relates to the technical field of nerve dissociation repair, in particular to an exosome-based nerve dissociation repair material EXO-BBC membrane and a preparation method thereof.

Background

Nerve dissociation is one of the common diseases in clinic, and a direct severed end suture technology or an autologous nerve transplantation method is generally adopted, but the limited donor source limits the application of the autologous nerve transplantation technology, so that a bridging material for replacing the autologous nerve has long become an important field of the current biomedical research. In recent years, researchers have proposed that biomaterials, cells, or neurotrophic factors are effective methods for repairing peripheral nerve interruptions. Among the above methods, an artificial nerve guide catheter-based surgery is considered as one of the most promising methods for treating peripheral nerve disconnection. The degradable biological conduit material is used for simulating the top end and the microstructure of a nerve and shows good thermal performance, and comprises materials such as synthetic polylactic acid, polycarbonate and biosynthetic cellulose. Among them, the Biosynthetic Cellulose (BC) is a biopolymer with excellent biocompatibility and purity, has low density and high porosity characteristics, and better biocompatibility, and has been used as a drug carrier to perform related application research in the biomedical field.

According to research reports, Exosomes (EXO) separated from adipose-derived Mesenchymal Stem Cells (MSCs) is a sub-cell double-layer membrane vesicle substance spitting from the inside to the outside of a cell, has the diameter of about 40-120nm, is cupulate or spherical under an electron microscope, contains proteins such as growth factors and cytokines similar to source cells and other active small molecular substances such as lipids, snRNA and miRNA, has the effects of increasing the number of myelofibers, promoting the formation of peripheral nerve myelin, further promoting the functional recovery after nerve injury and the like, and has potential application prospects in the field of nerve repair.

At present, the clinical peripheral nerve dissociation treatment materials are various, except for the autogenous nerve, the repair effect of other materials is not ideal, the price is high, and the safety risk is high. Therefore, there is a need to develop an economical and safe nerve dissociation repair material to meet the medical market demand.

Disclosure of Invention

In view of the above, the invention provides an exosome-based neuro-dissociation repair material EXO-BBC membrane, which solves the problems in the prior art.

The invention provides a preparation method of a nerve dissociation repair material EXO-BBC membrane based on exosomes, which comprises the following steps: soaking a freeze-dried mixed biosynthetic cellulose (BBC) membrane in normal saline at 25 ℃ for 5min, dividing the membrane into three layers by using forceps, drilling four holes at two ends of a single-layer BBC membrane (so as to carry out subsequent nerve dissociation repair operation), sterilizing the membrane at high temperature and high pressure (the subsequent operation is finished under the aseptic condition of 25 ℃), airing the water on the BBC membrane, placing the membrane in an aseptic six-hole plate, slowly and uniformly dripping 4 mu L of aseptic exosome solution onto the single-layer BBC membrane, and airing, wherein each mu L of exosome solution contains 1.9 multiplied by 10⁹And (3) carrying out exosome treatment to obtain an exosome-embedded nerve dissociation repair material EXO-BBC membrane, and storing the prepared EXO-BBC membrane under a 4-degree sterile condition for later use.

Preferably, the BBC film is prepared by the steps of:

(1) inoculating Gluconoacetobacter hansenii strain into Hestrin-Schramm (HS) modified culture medium, and statically culturing at 25 ℃ for 7 days to obtain original BC membrane;

(2) immersing the original BC membrane into 0.1M NaOH solution, stirring and washing for 24 hours at 50 ℃, and fully rinsing with distilled water to obtain a primary pure BC membrane;

(3) stirring the wet primary pure BC membrane by a homogenizer at the speed of 1000rpm until uniform cellulose pulp is obtained, filtering the uniform cellulose pulp by a filter under vacuum to form a thin paper-like hard membrane, and freeze-drying the thin paper-like hard membrane to obtain the BBC membrane. Preferably, the HS modified medium consists of 20.0g/L glucose, 5.0g/L peptone, 5.0g/L yeast extract, 2.7g/L disodium hydrogen phosphate and 1.5g/L citric acid.

Preferably, the exosome is prepared by the following steps:

(1) taking 4 th generation mouse adipose-derived Mesenchymal Stem Cells (MSCs) with good growth state, culturing for 72 hours by using a serum-free culture medium when the fusion degree reaches 90%, and collecting supernatant by using 8T 175 cell culture bottles;

(2) separating and purifying to obtain exosome: collecting the supernatant, centrifuging at 4 deg.C at 300 × g for 10 min, removing living cells, and collecting supernatant; centrifuging at 2000 Xg for 10 min at 4 deg.C to remove dead cells and leaving supernatant; centrifuging at 4 deg.C at 10000 Xg for 30 min, removing cell debris, and collecting supernatant; filtering the supernatant with 0.22 μm filter, concentrating the supernatant to 2mL with ultrafiltration tube, centrifuging at 100000 × g for 70 min, and collecting precipitate; the pellet was resuspended in 2mL PBS and aliquoted and stored at-80 deg.C until use.

The invention has the beneficial effects that:

on one hand, the invention provides a preparation method of an exosome-based nerve dissociation repair material EXO-BBC membrane, which is simple and easy and is convenient to popularize; on the other hand, the EXO-BBC membrane prepared by the method has good effect on repairing sciatic nerve amputation of rats, and the EXO-BBC membrane prepared by the method has the following discovery after evaluating the treatment effect on nerve regeneration by indexes such as gait, nerve function index, hot pain latency, nerve fiber regeneration at the amputation and the like: the EXO-BBC membrane has a remarkably better effect on the repair of sciatic nerve amputation of rats than an intramuscular injection exosome + BBC membrane or a BBC membrane used alone, and is safe and practical. Therefore, the EXO-BBC membrane prepared by the invention has wide clinical application prospect in the field of nerve dissociation repair.

Drawings

FIG. 1 is a morphological diagram of exosomes of example two under an electron microscope;

FIG. 2 is a graph of the gait blot at 8 weeks after the nerve bridge was dissociated in groups 4 of rats of the fifth example;

FIG. 3 is the neurological indices at 24 hours, 4 weeks and 8 weeks after the nerve bridge was detached in the group 4 rats of the fifth example;

FIG. 4 is the incubation period of thermal pain at 24 hours and 8 weeks after the nerve was disconnected in group 4 rats of example five;

FIG. 5 is a graph of nerve fiber distribution at week 8 after the nerve bridge was disconnected in groups 4 of rats of example five;

FIG. 6 is the number of nerve fibers at week 8 after the nerve bridge was disconnected in the group 4 rats of example five;

FIG. 7 is the mean nerve fiber diameter at 8 weeks after the nerve bridge was disconnected in groups 4 rats of example five.

Detailed Description

The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

The first embodiment is as follows: preparation of a BBC film comprising the steps of:

(1) inoculating Gluconoacetobacter hansenii strain into HS modified culture medium, and statically culturing at 25 ℃ for 7 days to obtain original BC membrane;

(3) stirring the wet primary pure BC membrane by a homogenizer at the speed of 1000rpm until uniform cellulose pulp is obtained, filtering the uniform cellulose pulp by a filter under vacuum to form a thin paper-like hard membrane, and freeze-drying the thin paper-like hard membrane to obtain the BBC membrane.

The observation by a scanning electron microscope, the specific surface area and the monofilament strength test show that: the porosity of the BBC film in the embodiment is 95%, the tensile strength reaches 15MPa, and the elastic modulus reaches 160 MPa.

Example two: the preparation of exosomes comprises the following steps:

The detection result of a Nanoparticle Tracking Analyzer (NTA) shows that the concentration of the exosome prepared in the test is 1.9 multiplied by 10¹¹mu.L/L. The exosome was tested by transmission electron microscopy, and the results are shown in fig. 1, where the exosome was irregular elliptical in shape with a diameter between 30-70 nm.

Example three: the preparation method of the exosome-based neuro-dissociation repair material EXO-BBC membrane comprises the following steps:

taking a freeze-dried BBC membrane (12 mm in length, 6mm in width and 0.3mm in thickness) of the example, soaking the BBC membrane in normal saline for 5 minutes at 25 ℃, dividing the BBC membrane into three layers by using forceps, punching four holes at two ends of a single-layer BBC membrane (so as to carry out subsequent nerve dissociation repair operation), sterilizing the BBC membrane at high temperature and high pressure (the subsequent operation is finished under the sterile condition of 25 ℃), drying the water on the BBC membrane, placing the BBC membrane in a sterile six-hole plate, and adding 4 mu L of sterile exosome solution (each mu L contains 1.9X 10⁹Individual exosomes) are slowly and evenly dripped on the single-layer BBC membrane and dried in the air to obtain the exosome-embedded neurolysis repair material EXO-BBC membrane. The prepared EXO-BBC membrane is stored under 4 ℃ aseptic condition for standby.

Example four: construction of rat sciatic nerve dissociation and repair model

Healthy adult male SD rats were divided into 4 groups of 6 rats each weighing 180-200 g. The experimental grouping was as follows:

EXO-BBC membrane group (group A): connecting the disconnected nerves by using an EXO-BBC membrane bridging method;

intramuscular injection of EXO + BBC membrane (group B): connecting the disconnected nerves by adopting a gastrocnemius injection exosome + BBC membrane bridging method;

BBC membrane group (group C): connecting the disconnected nerves by adopting a BBC membrane bridging method;

fascia group (group D): the severed nerves are connected using a method of fascia bridging.

Preoperative preparation (animal anesthesia and fixation sterilization): a1 ml syringe was used to inject 10% chloral hydrate (0.3ml/100g) into the abdominal cavity of a rat to anesthetize the rat, then the rat was fixed to a rat board in a prone position, the rat hair on the lower edge of the piriformis muscle of the right thigh was cut off by surgical scissors, and then the cut-off site was sterilized with alcohol 3 times.

Bridging surgery associated with BBC membranes: a diagonal incision was made in the right hip of the rat using a scalpel under sterile conditions, leaving the gluteal gap blunt and revealing the right sciatic nerve. After finding the sciatic nerve trunk, sewing two horns at one end of the membrane material on the sciatic nerve by using an 8-0 suture line, cutting off the sciatic nerve by using an ophthalmic scissors for 5mm, then sewing two horns at the other end of the membrane material on the other broken end of the sciatic nerve, wrapping and connecting the broken sciatic nerve, then using forceps to roll up the membrane material, sewing the middle part of the membrane, then immediately sewing a skin incision, and finally spraying alcohol for disinfection.

Administration: rats were fixed on a rat plate and 2. mu.L of the prepared exosomes (1.9X 10 per. mu.L) were aspirated by a 5. mu.L microinjector⁹Individual exosomes) were injected into the right gastrocnemius muscle of the rat, 5 minutes after the injection, the needle was left for 5 minutes to ensure complete absorption of the fluid, then slowly withdrawn, and another injection was performed 3 days later, with 4 μ L of exosomes co-injected twice.

Fascia bridging surgery: after finding the sciatic nerve trunk, fixing two ends of the sciatic nerve trunk by 8-0 suture, cutting off the sciatic nerve trunk with the diameter of 5mm by using an ophthalmic scissors after fixing, then taking a section of muscle tissue with the length of 5mm and the width of 2mm, suturing the muscle tissue and the nerve adventitia at the two ends of the sciatic nerve trunk by using 8-0 suture lines under an operation microscope, suturing skin incision, and spraying alcohol for disinfection.

Example five: evaluation of effect of EXO-BBC membrane applied to nerve dissociation repair

(1) Rat hindfoot gait detection and sciatic nerve functional index (SFI) test

Gait detection and SFI test methods: a long rat walking channel is self-made, and is 1m long, 15cm wide and 15cm high. And cutting white paper with the length of 1m and the width of 15cm, and paving the cut white paper at the bottom of the channel. After wearing canvas gloves, the rat tail is lifted up to expose double rear feet, carbon ink is brushed on the double rear feet of the rat by a brush, the rat is placed at one end of a walking channel, 4-5 footprints are left on each side of the rat in the process of creeping to the other end, and 3 variables (the length of the footprints, the width of toes and the distance of middle toes) of the test lateral foot (right lateral foot) and the normal lateral foot (left lateral foot) are selected for SFI scoring.

Gait analysis and blot assays were performed at 24 hours, 4 weeks, and 8 weeks after rat denervation (see fig. 2), respectively, and SFI values were calculated (see fig. 3). At 24 hours after surgery, the footprints of the rats in each group were unclear, the toes were completely indistinct, and the SFI values were almost indistinguishable. At week 4 after surgery, group A had a sharper footprint profile and more open interphalangeal extension than group B-D; the SFI values for group A were significantly better than those for group D-F (p < 0.05). Group a at postsurgical 8 weeks was significantly better than the other groups in gait, with a clearer footprint outline and more spread between the toes; group a outperformed group B in SFI values, significantly better than group C-D (p < 0.05).

The test results show that the effect of each group of bridging materials on the nerve dissociation repair is sequentially arranged from good to bad: EXO-BBC membrane group, intramuscular injection EXO + BBC membrane, BBC membrane group and fascia membrane group.

(2) Hot pain latency test

As shown in fig. 4, the results of the hot pain latency test indicate that: there was little difference in the hot pain latency at 24 hours post-surgery in the 4 groups of rats; the incubation period for thermal pain was significantly shorter in group a than in group B-D at week 8 (p < 0.01). This shows that the exosome-based neuro-ablative repair material EXO-BBC membrane of example three can significantly reduce the hot pain response time of rats and is the best neuro-ablative repair material in this experiment.

(3) Nerve fiber regeneration test

The observation result of the histology optical microscope is shown in fig. 5, the regenerated nerve fibers at the sciatic nerve disconnection position of the rats in the group A are more in number, more orderly in arrangement and thicker in myelin sheath at 8 weeks after the operation; the regenerated nerve fibers in the group B are distributed more uniformly and arranged regularly; the regenerated nerve fibers of the group C are not uniformly distributed and irregularly arranged; the regeneration quantity of the nerve fibers in the group D is very small, and the arrangement is very irregular. From the number of nerve fiber regeneration (fig. 6), group a was significantly better than groups B-D, and there were statistical differences (p < 0.01). While group a is also significantly superior to group B-D (p <0.01) in terms of nerve fiber diameter (fig. 7). These experimental results demonstrate that exosomal-based neuro-ablative repair material EXO-BBC membrane used in example three is the best neuro-ablative repair material in this experiment.

In conclusion, the indexes of gait, nerve function index, heat pain incubation period, nerve fiber regeneration at the scission part and the like show that the EXO-BBC membrane prepared by the invention has excellent effect on the repair of sciatic nerve of rats after sciatic nerve is dissociated, the repair effect is obviously superior to that of an intramuscular injection exosome and BBC membrane or that the BBC membrane is used alone, and the EXO-BBC membrane is an excellent nerve dissociation repair material and is safe and practical. Therefore, the EXO-BBC membrane prepared by the invention has wide clinical application prospect in the field of nerve dissociation repair.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A preparation method of a nerve dissociation repair material based on exosome is characterized by comprising the following steps: soaking a freeze-dried mixed biosynthetic cellulose membrane BBC in normal saline at 25 ℃ for 5min, dividing the BBC into three layers by using forceps, punching four holes at two ends of a single-layer BBC membrane, sterilizing the BBC membrane at high temperature and high pressure, drying the water on the BBC membrane in the air, placing the BBC membrane in a sterile six-hole plate, slowly and uniformly dripping 4 mu L of sterile exosome solution onto the single-layer BBC membrane, and drying the exosome-embedded neurolysis repair material in the air to obtain an exosome-embedded neurolysis repair material, wherein each mu L of exosome solution contains 1.9 multiplied by 10⁹And (4) one exosome.

2. The method for preparing an exosome-based neuro-ionization repair material according to claim 1, wherein the BBC membrane is prepared by the following steps:

(3) and (3) stirring the primary pure BC membrane by a homogenizer at the speed of 1000rpm until uniform cellulose pulp is obtained, filtering the uniform cellulose pulp by a filter under vacuum to form a thin paper-like hard membrane, and freeze-drying to obtain the BBC membrane.

3. The method for preparing an exosome-based neuro-ionization repair material according to claim 2, wherein the HS-modified medium consists of 20.0g/L glucose, 5.0g/L peptone, 5.0g/L yeast extract, 2.7g/L disodium hydrogenphosphate and 1.5g/L citric acid.

4. The method for preparing an exosome-based neuro-dissociation repair material according to claim 1, wherein the exosome is prepared by the following steps:

(1) taking the 4 th generation of mouse adipose-derived mesenchymal stem cells with good growth state, culturing the mouse adipose-derived mesenchymal stem cells for 72 hours by using a serum-free culture medium when the fusion degree reaches 90%, and collecting the supernatant by using 8T 175 cell culture bottles;

5. An exosome-based neuro-ablative repair material prepared according to the method of any one of claims 1-4.