Preparation and application of novel bioadhesive with high bioadhesion and high biocompatibility for promoting peripheral nerve injury repair

Abstract

The application belongs to the field of medicine and health technology, and particularly relates to preparation and application of a novel bioadhesive with high biological adhesion and high biological compatibility for promoting peripheral nerve injury repair, which comprises the following steps: (1) grafting acetylcysteine and dopamine on chitosan to prepare modified chitosan gel with self-crosslinking performance as an adhesive layer; (2) preparing sodium alginate and polyacrylamide covalent / non-covalent double crosslinked gel with high strength and toughness as a substrate layer; and (3) uniformly coating the adhesive layer on the substrate layer, and cutting the adhesive layer into blocks of different sizes according to the requirements of use, so that the novel bioadhesive with high biological adhesion and high biological compatibility for promoting peripheral nerve injury repair is obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the preparation of a new type of bioadhesive with high bioadhesion and high biocompatibility, and its application in peripheral nerve injury repair, belonging to the field of medicine and health technology. BACKGROUND

[0002] Only a part of the patients with peripheral nerve injury can be completely cured, and most of the patients will still suffer from sequelae, including dysfunction, after the treatment is completed. Therefore, improving the repair effect of peripheral nerve injury is of great significance to patients with nerve injury.

[0003] At present, the repair methods of peripheral nerve injury mainly include: (1) surgical method (including end-to-end suture, nerve transplantation, etc.); (2) tissue engineering method (including conduit stent method and biological adhesion method); (3) gene engineering method; (4) nerve growth factor regulation method, etc. Because the suture has adverse effects on the recovery of nerve function, it also increases the probability of nerve scar formation. At present, the tissue engineering method has become one of the popular research directions instead of surgical method. The conduit stent usually needs a recovery period of 4-6 months, and the cost is high; the mechanical strength of the marketed fibrin glue in the biological adhesion method is poor. The biological adhesion hydrogel used for soft tissue repair can simplify the surgical process, reduce the damage to the tissue, and to some extent, can replace the suture. Although there is no clinical case report, the biological adhesives similar to fibrin glue have become a popular research field.

[0004] The existing literature on the biological adhesives for peripheral nerve injury repair still has certain defects and deficiencies, such as: adding oxidizing agents, surfactants or strong alkalis and other cross-linking agents to increase the strength of the gel, which reduces the biological safety of the gel; the synthesis method is complex or the cost is high; the degradation time does not meet the peripheral nerve injury repair cycle, etc.

[0005] In view of the problems existing in the current peripheral nerve injury repair method, the ideal biological adhesives for peripheral nerve injury repair should have the functions of reducing the adverse effects of suture, good biological adhesion and biocompatibility, simple preparation and use, low cost, and no need for additional additives.

[0006] Chitosan is the only natural alkaline nitrogen-containing polysaccharide. After modification, it can be prepared into nanometer particles, hydrogel, film and other forms of materials, which are widely used in biomedical fields such as drug carriers, tissue engineering, medical dressings and so on. Chitosan and its derivatives have the following advantages as peripheral nerve repair materials: ① non-toxic, low irritability, biodegradable and good biocompatibility; ② chitosan can promote cell proliferation, inhibit fibroblast proliferation and reduce scar tissue formation; ③ chitosan has low cost and high modification potential; ④ chitosan oligosaccharide (COS), the degradation product of chitosan, can promote cell proliferation and prevent exogenous injury-induced apoptosis; and so on.

[0007] According to the performance requirements of the bioadhesive for peripheral nerve injury repair, a new type of bioadhesive is designed, which is composed of an adhesive layer and a substrate layer. The adhesive layer is a hydrogel with good bioadhesion, which can strongly adhere to the tissue; the substrate layer is a gel film material, which plays multiple functions: ① covering the adhesive layer, facilitating the operation of wrapping the nerve, and allowing the adhesive layer to adhere tightly and firmly to the nerve tissue; ② having strong toughness, which can resist external additional tension and tensile force, preventing the separation of the adhered nerve.

[0008] A thiolated chitosan gel (CS-NAC-DA) coupled with acetylcysteine (NAC) and dopamine (DA) is prepared as the adhesive layer by coupling on the chitosan backbone molecule. CS-NAC-DA has the following characteristics: ① it can undergo self-crosslinking reaction at a certain concentration without the need for additional crosslinking agent, which enhances the bioadhesion while maintaining good biocompatibility; ② as a hydrogel, it can uniformly cover the injured tissue, and due to the introduction of dopamine and thiol groups, it has good bioadhesion, which can adhere the injured nerve ends together under physiological conditions.

[0009] A non-covalent / covalent double-crosslinked alginate and polyacrylamide gel is prepared as the substrate layer. The non-covalently crosslinked alginate gel can play a role in delaying and dissipating the external influence so that it cannot directly act on the injured site; the covalently crosslinked polyacrylamide gel has high strength. The substrate layer has high biocompatibility, high toughness and high energy dissipation, which can help the adhesive layer to maintain stability in the complex body environment and continuously play a role in assisting repair.

[0010] The adhesive layer is uniformly coated on the substrate layer, and cut into different sizes according to the requirements of use, i.e. to obtain the designed new type of bioadhesive. The substrate layer can also form hydrogen bonds with the adhesive layer, and the two can adapt to each other and work synergistically.

[0011] In summary, the new biological adhesive can be used more conveniently in peripheral nerve injury repair by combining the adhesive layer and the base layer, and the material strength is sufficient to meet the needs of protecting the damaged nerves, and has the prospects of industrialized preparation and clinical transformation. Meanwhile, the new biological adhesive can be explored in more fields due to its excellent properties, such as blood vessels, cartilage, joints and other non-medical fields, and can become the preferred material in future tissue engineering. SUMMARY

[0012] The purpose of the present application is to provide a new biological adhesive for repairing peripheral nerve injury in view of the defects of the commercially available fibrin glue and the existing research.

[0013] The adhesive layer is prepared by coupling acetylcysteine and dopamine on the molecular chain of chitosan; the base layer is prepared by covalent / non-covalent double crosslinking of sodium alginate and polyacrylamide; and the new biological adhesive with mechanical strength and biocompatibility is prepared by uniformly coating the adhesive layer on the base layer.

[0014] The new biological adhesive provided by the present application has the following preparation process:

[0015] (1) Preparation of acetylcysteine-coupled mercaptized chitosan (CS-NAC):

[0016] (2) Preparation of dopamine and acetylcysteine-coupled mercaptized chitosan (CS-NAC-DA):

[0017] (3) Preparation of the base layer:

[0018] (4) Preparation of the new biological adhesive:

[0019] The new biological adhesive is obtained by spreading an appropriate amount of CS-NAC-DA on the surface of the base layer.

[0020] In detail, the new biological adhesive provided by the present application has the following preparation process:

[0021] (1) Preparation of acetylcysteine-coupled mercaptized chitosan (CS-NAC):

[0022] (1.1) Acidification and dissolution of chitosan (CS): chitosan is weighed and placed in a triangular flask, hydrochloric acid solution is added, and stirring is performed to fully swell and dissolve the chitosan, thereby obtaining a chitosan solution;

[0023] (1.2) Preparation of acetylcysteine (NAC) solution: solid acetylcysteine is weighed and placed in a triangular flask, deionized water is added, and the solution is dissolved at room temperature for standby;

[0024] (1.3) Preparation of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) solution: EDC and NHS were weighed into a flask, deionized water was added, and the solution was dissolved at room temperature and used as prepared;

[0025] (1.4) Carboxyl activation: the EDC and NHS solution was slowly added dropwise to the stirring NAC solution, and after the dropwise addition was completed, the reaction was stirred at room temperature to activate the carboxyl groups;

[0026] (1.5) Coupling reaction and pH adjustment: the chitosan solution was slowly added dropwise to the stirring mixture of EDC, NHS and NAC, and after the dropwise addition was completed, the stirring was continued at room temperature, and the pH value was adjusted;

[0027] (1.6) Dialysis and freeze-drying: after stirring, the reaction solution was placed in a dialysis bag, dialyzed with deionized water, and after dialysis was completed, freeze-drying was performed to obtain CS-NAC;

[0028] (2) Preparation of thiolated chitosan coupled with dopamine and acetylcysteine (CS-NAC-DA):

[0029] (2.1) CS-NAC dissolution: CS-NAC was weighed into a flask, deionized water was added, and the solution was stirred to swell;

[0030] (2.2) Preparation of dopamine (DA) solution: DA solid was weighed into a flask, deionized water was added, and the solution was dissolved at room temperature and used as prepared;

[0031] (2.3) Preparation of EDC and NHS solution: EDC and NHS were weighed into a flask, deionized water was added, and the solution was dissolved at room temperature and used as prepared;

[0032] (2.4) Carboxyl activation: the EDC and NHS solution was slowly added dropwise to the stirring DA solution, and after the dropwise addition was completed, the reaction was stirred at room temperature to activate the carboxyl groups;

[0033] (2.5) Coupling reaction and pH adjustment: the CS-NAC solution was slowly added dropwise to the stirring mixture of EDC, NHS and DA, and after the dropwise addition was completed, the stirring was continued at room temperature, and the pH value was adjusted, and the mixture solution was placed in a chromatography cabinet and stirred;

[0034] (2.6) Dialysis and freeze-drying: the reaction solution was placed in a dialysis bag, dialyzed with deionized water, and after dialysis was completed, freeze-drying was performed to obtain CS-NAC-DA;

[0035] (3) Preparation of base layer:

[0036] (3.1) Sodium alginate solution and acrylamide solution prepared with HBSS buffer solution, stirred on a magnetic stirrer until all the solids were dissolved, and left to remove its bubbles;

[0037] (3.2) Prepare appropriate amount of methylene bisacrylamide (MBAA) solution, calcium sulfate (CaSO4) solution, ammonium persulfate (APS) solution with HBSS buffer solution;

[0038] (3.3) Add MBAA solution, tetramethyl ethylenediamine (TEMED) and CaSO4 solution into the mixed solution of sodium alginate and acrylamide under high-speed stirring, and stir until mixed evenly;

[0039] (3.4) Add APS solution into the mixed solution quickly, and stir quickly;

[0040] (3.5) Pour the mixed solution into the mold, and leave it overnight to crosslink, and the base layer is obtained;

[0041] (4) Preparation of the new biological adhesive:

[0042] Use a syringe to suck the appropriate amount of CS-NAC-DA gel, and slowly spread it on the surface of the base layer to obtain the new biological adhesive.

[0043] Preferably, (1) preparation of acetylcysteine-coupled mercaptized chitosan (CS-NAC):

[0044] Chitosan (CS) acidification and dissolution: weigh 250-1000 mg of chitosan into a triangular flask, add hydrochloric acid solution, and stir to fully swell and dissolve to obtain a chitosan solution;

[0045] Prepare acetylcysteine (NAC) solution: weigh 500-2000 mg of solid acetylcysteine into a triangular flask, add deionized water, and dissolve at room temperature for standby;

[0046] Prepare EDC and NHS solution: weigh 500-2000 mg of EDC and 250-1000 mg of NHS into a triangular flask, add deionized water, and dissolve at room temperature for standby;

[0047] Carboxyl activation: slowly add the EDC and NHS solution into the stirring NAC solution, and after the addition is completed, stir at room temperature to carry out carboxyl activation;

[0048] Coupling reaction and pH adjustment: slowly add the chitosan solution into the mixed solution of EDC, NHS and NAC under stirring, after the addition is completed, continue to stir at room temperature, and then adjust the pH to 5.8-6.0 by adding sodium hydroxide solution, and place the mixed solution in a chromatography cabinet for stirring;

[0049] Dialysis and freeze-drying: After stirring, the reaction solution was loaded into dialysis bags, dialyzed with deionized water, and freeze-dried after dialysis to obtain CS-NAC.

[0050] Preferably, (2) preparation of thiolated chitosan coupled with dopamine and acetylcysteine (CS-NAC-DA):

[0051] CS-NAC dissolution: 250-1000 mg of CS-NAC was weighed into a triangular flask and deionized water was added. The solution was stirred to swell completely;

[0052] Preparation of dopamine (DA) solution: 750-3000 mg of DA solid was weighed into a triangular flask and deionized water was added. The solution was dissolved at room temperature and ready for use;

[0053] Preparation of EDC and NHS solution: 750-3000 mg of EDC and 300-1500 mg of NHS were weighed into a triangular flask and deionized water was added. The solution was dissolved at room temperature and ready for use;

[0054] Carboxyl activation: The EDC and NHS solution was drawn out and slowly added dropwise to the stirring DA solution. After the dropwise addition was completed, the carboxyl activation reaction was carried out at room temperature under stirring;

[0055] Coupling reaction and pH adjustment: The CS-NAC solution was slowly added dropwise to the stirring mixed solution of EDC, NHS and DA. After the dropwise addition was completed, the solution was stirred at room temperature. After the dropwise addition was completed, the stirring was continued at room temperature. Sodium hydroxide solution was added dropwise to adjust the pH to 5.8-6.0, and then the stirring was stopped. The mixed solution was placed in a chromatography cabinet for stirring;

[0056] Dialysis and freeze-drying: The reaction solution was loaded into dialysis bags, dialyzed with deionized water, and freeze-dried after dialysis to obtain CS-NAC-DA.

[0057] Preferably, (3) preparation of the base layer:

[0058] First step: 1-4% sodium alginate solution and 6-24% acrylamide solution were prepared using HBSS buffer and stirred on a magnetic stirrer until the solids were completely dissolved. The solution was left to stand to remove the air bubbles;

[0059] Second step: An appropriate amount of 1-3% methylene bisacrylamide (MBAA) solution, 0.1-0.75 M calcium sulfate (CaSO4) solution, and 0.1-0.5 M ammonium persulfate (APS) solution were prepared using HBSS buffer;

[0060] Step 3: Add 36-140 μL MBAA solution, 8-32 μL tetramethylethylenediamine (TEMED) and 200-400 μL CaSO4 solution dropwise to the sodium alginate and acrylamide mixed solution under high-speed stirring, and stir until the mixture is homogeneous.

[0061] Step 4: Quickly add 250-1000 μL of APS solution to the mixed solution and stir rapidly;

[0062] Step 5: Pour the mixed solution into the mold and let it stand overnight to allow it to crosslink, thus obtaining the base layer.

[0063] Preferably, (4) the preparation of novel bioadhesives:

[0064] Prepare 2-8% CS-NAC-DA gel, use a syringe to slowly drip an appropriate amount onto the surface of the basal layer, and spread it evenly. Cut it into appropriate sizes as needed to obtain a novel bioadhesive.

[0065] More preferably, the preparation of the novel bio-adhesive of the present invention includes the following steps:

[0066] (1) Preparation of acetylcysteine-coupled thiolated chitosan (CS-NAC):

[0067] Chitosan (CS) acidification and dissolution: Weigh 500 mg of chitosan with a molecular weight of 1000 kDa and place it in a 100 mL Erlenmeyer flask. Add 30 mL of hydrochloric acid solution with pH = 5 and stir for 2 h to allow it to swell fully.

[0068] Preparation of acetylcysteine ​​(NAC) solution: Weigh 1000 mg of solid acetylcysteine ​​into a 50 mL Erlenmeyer flask, add 20 mL of deionized water, dissolve at room temperature, and set aside.

[0069] Preparation of EDC and NHS solution: Weigh 1000 mg of EDC and 500 mg of NHS into a 50 mL Erlenmeyer flask, add 10 mL of deionized water, dissolve at room temperature, and set aside.

[0070] Carboxyl activation: EDC and NHS solution were slowly added dropwise to NAC solution under stirring. After the addition was complete, the mixture was stirred at room temperature for half an hour to activate the carboxyl group.

[0071] Coupling reaction and pH adjustment: Chitosan solution was slowly added dropwise to a mixed solution of EDC, NHS, and NAC under stirring. After the addition was complete, stirring was continued at room temperature for half an hour. Then, the pH was adjusted by adding 1M sodium hydroxide solution dropwise, and the reaction solution and pH meter count were observed. Once the count stabilized at 5.8-6.0, the addition of sodium hydroxide solution was stopped, and the mixed solution was placed in a chromatography cabinet at 4℃ and stirred for 8 hours.

[0072] Dialysis and freeze-drying: After stirring, the reaction solution was loaded into a dialysis bag with a molecular weight cut-off of 8000-14000 Da, dialyzed with 3 L of deionized water for more than 6 times, and each dialysis interval was 6-8 h. After dialysis, freeze-drying was performed, and CS-NAC was obtained. The freeze-dried product was sealed and stored in a refrigerator at 4°C for standby use.

[0073] (2) Preparation of thiolated chitosan coupled with dopamine and acetylcysteine (CS-NAC-DA):

[0074] Dissolution of CS-NAC: 500 mg of CS-NAC was weighed and placed in a 200 mL triangular flask, 50 mL of deionized water solution was added, and stirring was performed for 1 h to fully swell.

[0075] Preparation of dopamine (DA) solution: 1500 mg of DA solid was weighed and placed in a 50 mL triangular flask, 50 mL of deionized water was added, and it was dissolved at room temperature for standby use.

[0076] Preparation of EDC and NHS solution: 1500 mg of EDC and 750 mg of NHS were weighed and placed in a 50 mL triangular flask, 20 mL of deionized water was added, and it was dissolved at room temperature for standby use.

[0077] Carboxyl activation: The EDC and NHS solution was sucked out and slowly added dropwise to the stirring DA solution. After the addition was completed, carboxyl activation was performed at room temperature for half an hour.

[0078] Coupling reaction and pH adjustment: The CS-NAC solution was slowly added dropwise to the mixed solution of EDC, NHS and DA under stirring. After the addition was completed, stirring was continued at room temperature for half an hour. After the addition was completed, 1 M sodium hydroxide solution was added dropwise to adjust the pH, and the reaction solution and pH meter count were observed. After the count was stable, the addition of sodium hydroxide solution was stopped when the pH reached 5.8-6.0, and the mixed solution was placed in a chromatography cabinet at 4°C for stirring for 48 h.

[0079] Dialysis and freeze-drying: The reaction solution was loaded into a dialysis bag with a molecular weight cut-off of 8000-14000 Da, dialyzed with 3 L of deionized water for more than 6 times, and each dialysis interval was 6-8 h. After dialysis, freeze-drying was performed, and CS-NAC-DA was obtained. The freeze-dried product was sealed and stored in a refrigerator at 4°C for standby use.

[0080] (3) Preparation of the base layer:

[0081] First step: 2% sodium alginate (viscosity: 20-100 Cp) solution and 12% acrylamide solution 20 mL were prepared using HBSS buffer, stirred uniformly on a magnetic stirrer until the solids were completely dissolved, and the bubbles were removed by standing;

[0082] Second step: prepare 2% methylene bisacrylamide (MBAA) solution, 0.375M calcium sulfate (CaSO4) solution, 0.27M ammonium persulfate (APS) solution using HBSS buffer solution;

[0083] Third step: add 72μL MBAA solution, 16μL tetramethyl ethylenediamine (TEMED) and 400μL CaSO4 solution into the sodium alginate and acrylamide mixed solution in high speed stirring one by one drop by drop, and stir until mixed evenly;

[0084] Fourth step: quickly add 500μL APS solution into the mixed solution, and stir quickly for 2 minutes;

[0085] Fifth step: pour the mixed solution into the mold, and place it overnight to crosslink, thereby obtaining the base layer.

[0086] (4) Preparation of the novel biological adhesive:

[0087] Prepare 4% CS-NAC-DA gel, use a 1mL syringe to take an appropriate amount and slowly drop it on the surface of the base layer, and make it spread evenly (about 10μL / 0.32cm 2 ), and cut it into appropriate size according to the need, thereby obtaining the novel biological adhesive.

[0088] Among them, the preparation process of 4% CS-NAC-DA gel is as follows:

[0089] First step: weigh 120mg of CS-NAC-DA freeze-dried product, and put it into a Westlin bottle;

[0090] Second step: use a pipette to take 3ml of deionized water and drop it into the Westlin bottle, so that the freeze-dried product swells for 2 hours;

[0091] Third step: vortex the swelled 4% CS-NAC-DA to mix evenly, and stand to remove bubbles.

[0092] Another object of the present application is to provide the novel biological adhesive as a medical material for preventing adhesion after peripheral nerve injury repair surgery.

[0093] Another object of the present application is to provide the application of the novel biological adhesive in the preparation of drugs for promoting peripheral nerve injury repair, tendon adhesion, blood vessel injury and surgical incision.

[0094] The novel biological adhesive described in the present application can be used in combination with other pharmaceutical active ingredients, and the other pharmaceutical active ingredients are added to the biological adhesive.

[0095] Among them, the other pharmaceutical active ingredients are selected from:

[0096] (1) Growth factors such as nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), myofibroblast growth factor (MGF), etc.

[0097] (2) Drugs such as gangliosides, citicoline, etc.

[0098] Compared with the existing biological adhesives, the present application has the following advantages:

[0099] (1) Good biocompatibility, biodegradable and suitable degradation rate; (2) The adhesive layer has self-crosslinking property and can form gel by self-crosslinking without additional crosslinking agent, which is convenient for preparation and use and does not affect the biological safety; (3) The adhesive layer has sufficient biological adhesion, which can firmly cover the moist and small nerve and adhere the nerve end to promote the repair of nerve injury; (4) The substrate layer has strong toughness and high strength, which can greatly reduce the influence of external force on the damaged part and has good biological adhesion and is not easy to fall off from the surgical site; (5) All raw materials are easy to obtain and the cost is low.

[0100] Compared with the prior art, the biggest technical improvement and innovation of the present application lies in steps (2) and (4). In the prior art, the finished adhesive generally needs to be treated by oxidation, alkalization and the like. This treatment step increases the use complexity, reduces the biocompatibility and has weak anti-interference ability. The innovation points of the present application are that ① the finished CS-NAC-DA has self-crosslinking property and does not need additional gel forming step; and ② the damaged nerve part is adhered by using the substrate layer to load the adhesive layer instead of using the traditional treatment method. This method ensures the high biological compatibility of the adhesive layer, and the high toughness of the substrate layer can reduce the interference to ensure the effectiveness of the adhesive layer.

[0101] The present application also has the following advantages: the adhesive layer has self-crosslinking property and does not need to use additional crosslinking agent; the substrate layer has strong toughness and the function of protecting the surrounding nerve injury; the new biological adhesive has excellent mechanical properties, strong biological adhesion and high biological compatibility, and can promote the repair process of peripheral nerve injury, which is obviously superior to the conventional surgical method in terms of repair effect.

[0102] The reaction conditions of the present application are obtained after a large number of screening, and the amount of CS, NAC, DA and other components, solution concentration, reaction time, pH value, reaction temperature and other conditions are optimized. BRIEF DESCRIPTION OF DRAWINGS

[0103] Figure 1 , CS-NAC different feeding ratio-mercapto coupling rate graph

[0104] Figure 2 , CS-NAC different reaction time-mercapto coupling rate graph

[0105] Figure 3 Figure 6, CS-NAC-DA different feeding amount-dopamine coupling rate graph

[0106] Figure 4 Figure 7, CS-NAC-DA different reaction time-dopamine coupling rate graph

[0107] Figure 5 Figure 8, Adhesive layer gel material CS-NAC-DA ultraviolet spectrum

[0108] Figure 6 Figure 9, Adhesive layer gel material scanning electron microscope image

[0109] Figure 7 Figure 10, Rheological amplitude scanning spectrum of different concentrations of CS-NAC-DA gel

[0110] Figure 8 Figure 11, Rheological frequency scanning spectrum of different concentrations of CS-NAC-DA gel

[0111] Figure 9 Figure 12, Rheological time scanning spectrum of different concentrations of CS-NAC-DA gel

[0112] Figure 10 Figure 13, Rheological stress-strain test spectrum of different concentrations of CS-NAC-DA gel

[0113] Figure 11 Figure 14, Bioadhesion strength of different concentrations of CS-NAC-DA gel

[0114] Wherein **** represents P<0.0001 compared with fibrin glue; ## represents P<0.01 compared with 4% CS-NAC-DA; #### represents P<0.0001 compared with 4% CS-NAC-DA. The picture shows the average number, and the error line indicates ±SD.

[0115] Figure 12 Figure 15, Substrate layer scanning electron microscope image

[0116] Figure 13 Figure 16, Rheological frequency scanning spectrum of substrate layer

[0117] Figure 14 Figure 17, Rheological stress-strain test spectrum of substrate layer

[0118] Figure 15 Figure 18, Remaining mass change curve of adhesive layer in vitro degradation over time

[0119] Figure 16 Figure 19, Remaining mass change curve of substrate layer in vitro degradation over time

[0120] Figure 17Comparison of the remaining mass and percentage of different parts of the new biological adhesive in vivo degradation experiment per week

[0121] Figure 18 Figure of cytotoxicity of gel extract on L929 cells.

[0122] a: 100% CS-NAC-DA extract group; b: 75% CS-NAC-DA extract group; c: 50% CS-NAC-DA extract group; d: 25% CS-NAC-DA extract group; e: 100% base layer extract group; f: 75% base layer extract group; g: 50% base layer extract group; h: 25% base layer extract group.

[0123] Figure 19 Figure of the change of the rats' stride length over time after the sciatic nerve transection repair surgery.

[0124] Wherein, A is the control group; B is the transection group; C is the positive control group; D is the surgery group; E is the experimental group one; F is the experimental group two. The test method adopts one-way ANOVA, * represents P<0.05 compared with the transection group; # represents P<0.05 compared with the positive control group; = represents P<0.05 compared with the surgery group; + represents P<0.05 compared with the experimental group one.

[0125] Figure 20 Figure of the change of the rats' grip strength over time after the sciatic nerve transection repair surgery.

[0126] Wherein, A is the control group; B is the transection group; C is the positive control group; D is the surgery group; E is the experimental group one; F is the experimental group two. * represents P<0.05 compared with the transection group; # represents P<0.05 compared with the positive control group; = represents P<0.05 compared with the surgery group; + represents P<0.05 compared with the experimental group one.

[0127] Figure 21 Measurement of the rats' sciatic nerve pull-out force after the sciatic nerve transection repair surgery for six weeks.

[0128] Wherein, A is the control group; B is the transection group; C is the positive control group; D is the surgery group; E is the experimental group one; F is the experimental group two. * represents P<0.05 compared with the transection group; # represents P<0.05 compared with the positive control group; = represents P<0.05 compared with the surgery group; + represents P<0.05 compared with the experimental group one.

[0129] Figure 22IL-6 content determination of rats after sciatic nerve transection repair surgery for six weeks. Among them, A is the control group; B is the transection group; C is the positive control group; D is the surgery group; E is the experimental group one; F is the experimental group two. * represents P < 0.05 compared with the control group; ** represents P < 0.01 compared with the control group.

[0130] Figure 23 The curve graph of the stride of rats changing with time after rat Achilles tendon transection repair surgery. Among them, A is the control group; B is the transection group; C is the surgery group one; D is the surgery group two; E is the experimental group one; F is the experimental group two. * represents P < 0.05 compared with the transection group; # represents P < 0.05 compared with the surgery group two; = represents P < 0.05 compared with the experimental group one.

[0131] Figure 24 The curve graph of the grip strength of rats changing with time after rat Achilles tendon transection repair surgery. Among them, A is the control group; B is the transection group; C is the surgery group one; D is the surgery group two; E is the experimental group one; F is the experimental group two. * represents P < 0.05 compared with the transection group; # represents P < 0.05 compared with the surgery group two; = represents P < 0.05 compared with the experimental group one.

[0132] Figure 25 Sciatic nerve pull-out force determination of rats after rat Achilles tendon transection repair surgery for two weeks. Among them, A is the control group; B is the transection group; C is the surgery group one; D is the surgery group two; E is the experimental group one; F is the experimental group two. # represents P < 0.05 compared with the surgery group two; = represents P < 0.05 compared with the experimental group one.

[0133] Figure 26 Sciatic nerve pull-out force determination of rats after rat Achilles tendon transection repair surgery for four weeks. Among them, A is the control group; B is the transection group; C is the surgery group one; D is the surgery group two; E is the experimental group one; F is the experimental group two. * represents P < 0.05 compared with the transection group; # represents P < 0.05 compared with the surgery group two; = represents P < 0.05 compared with the experimental group one.

[0134] Figure 27 IL-6 content determination of rats after rat Achilles tendon transection repair surgery for two weeks. Among them, A is the control group; B is the transection group; C is the surgery group one; D is the surgery group two; E is the experimental group one; F is the experimental group two. # represents P < 0.05 compared with the control group; * represents P < 0.05 compared with the transection group; = represents P < 0.05 compared with the experimental group one.

[0135] Figure 28The IL-6 content of rats was determined four weeks after the rat achilles tendon transection repair operation. Among them, A is the control group; B is the transection group; C is the operation group one; D is the operation group two; E is the experimental group one; F is the experimental group two. # represents compared with the control group, P<0.05; * represents compared with the transection group, P<0.05; = represents compared with the experimental group one, P<0.05. DETAILED DESCRIPTION

[0136] The present application is further explained and described by the following specific examples, but is not used as a limitation to the present application.

[0137] Example 1, synthesis of adhesive layer material

[0138] The synthesis method of acetylcysteine-coupled thiolated chitosan is as follows:

[0139] Chitosan acidification and dissolution: 500 mg of chitosan with a molecular weight of 1000 KD was placed in a 100 mL triangular flask, 30 mL of hydrochloric acid solution with pH = 5 was added, and it was stirred for 2 h to make it fully swollen.

[0140] Preparation of acetylcysteine solution: 1000 mg of solid acetylcysteine was weighed into a 50 mL triangular flask, 20 mL of deionized water was added, and it was dissolved at room temperature for standby.

[0141] Preparation of EDC and NHS solution: 1000 mg of EDC and 500 mg of NHS were placed in a 50 mL triangular flask, 10 mL of deionized water was added, and it was dissolved at room temperature for standby.

[0142] Carboxyl activation: slowly add the EDC and NHS solution drop by drop to the stirring NAC solution, after the dropwise addition is completed, stir at room temperature for half an hour for carboxyl activation.

[0143] Coupling reaction and pH adjustment: slowly add the chitosan solution drop by drop to the stirring mixed solution of EDC, NHS and NAC, after the dropwise addition is completed, continue to stir at room temperature for half an hour. Then adjust the pH by dropwise adding 1M sodium hydroxide solution, and observe the reaction solution and the pH meter count, stop adding sodium hydroxide solution when the count is stable at 5.8-6.0, and place the mixed solution in a 4℃ chromatography cabinet for stirring for 8 h.

[0144] Dialysis and freeze-drying: after stirring, the reaction solution was loaded into a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and dialyzed with 3L deionized water for more than 6 times, with an interval of 6-8 h each time. After dialysis, freeze-drying, the freeze-dried product was sealed and stored in a 4℃ refrigerator for standby.

[0145] The synthesis method of acetylcysteine-coupled thiolated chitosan is as follows:

[0146] Thiol chitosan dissolution: 500 mg of thiol chitosan was weighed into a 200 mL flask, 50 mL of deionized water was added, and it was stirred for 1 h to fully swell.

[0147] Dopa solution preparation: 1500 mg of Dopa solid was weighed into a 50 mL flask, 50 mL of deionized water was added, and it was dissolved at room temperature and set aside.

[0148] EDC and NHS solution preparation: 1500 mg of EDC and 750 mg of NHS were weighed into a 50 mL flask, 20 mL of deionized water was added, and it was dissolved at room temperature and set aside.

[0149] Carboxyl activation: The EDC and NHS solution was aspirated and slowly added dropwise to the stirring Dopa solution. After the dropwise addition was complete, the carboxyl activation was carried out at room temperature for half an hour with stirring.

[0150] Coupling reaction and pH adjustment: The thiol chitosan solution was slowly added dropwise to the stirring EDC, NHS, and DA mixed solution. After the dropwise addition was complete, the solution was stirred at room temperature for half an hour. Then, 1 M sodium hydroxide solution was added dropwise to adjust the pH, and the reaction solution and pH meter count were observed. After the count was stable, the dropwise addition of sodium hydroxide solution was stopped when the pH reached 5.8-6.0, and the mixed solution was placed in a 4°C chromatography cabinet and stirred for 48 h.

[0151] Dialysis and freeze-drying: The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and dialyzed with 3 L of deionized water for more than 6 times, with an interval of 6-8 h between each dialysis. After dialysis, it was freeze-dried, and the freeze-dried product was sealed and stored in a 4°C refrigerator for use.

[0152] Example 2, optimization of the preparation process of the adhesive layer material

[0153] CS-NAC feed quantity optimization

[0154] Chitosan acidification and dissolution: 100 mg of chitosan with a molecular weight of 1000 KD was weighed into a 50 mL flask, 20 mL of pH 5 hydrochloric acid solution was added, and it was stirred for 1 h to fully swell, and four groups were carried out in parallel.

[0155] Acetylcysteine solution preparation: 100 mg (group A), 150 mg (group B), 200 mg (group C), and 300 mg (group D) of acetylcysteine solid were weighed into 50 mL flasks, respectively, 10 mL of deionized water was added, and it was dissolved at room temperature and set aside.

[0156] Preparation of EDC and NHS solution: 100 mg of EDC and 50 mg of NHS (group A), 150 mg of EDC and 75 mg of NHS (group B), 200 mg of EDC and 100 mg of NHS (group C), and 300 mg of EDC and 150 mg of NHS (group D) were weighed into 50 mL triangular flasks, respectively, and 10 mL of deionized water was added. The solutions were dissolved at room temperature and were ready for use.

[0157] Carboxyl activation: The EDC and NHS solution of the same group was slowly added dropwise to the stirring NAC solution. After the addition was completed, the reaction was stirred at room temperature for half an hour to activate the carboxyl group.

[0158] Coupling reaction and pH adjustment: The chitosan solution was slowly added dropwise to the stirring mixture of EDC, NHS and NAC. After the addition was completed, the reaction was continued to stir at room temperature for half an hour. Then, 1 M sodium hydroxide solution was added dropwise to adjust the pH. The reaction solution and pH meter were observed. When the count was stable, the addition of sodium hydroxide solution was stopped when the pH reached 5.8-6.0. The mixture was placed in a 4°C chromatography cabinet and stirred for 8 h.

[0159] Dialysis and freeze-drying: After stirring, the reaction solution was loaded into a dialysis bag with a molecular weight cut-off of 8000-14000 Da. The dialysis was performed for more than 6 times with 3 L of deionized water, and the interval between each dialysis was 6-8 h. After dialysis, freeze-drying was performed. The freeze-dried product was sealed and stored in a 4°C refrigerator for future use.

[0160] The thiol coupling rate of the freeze-dried products of groups A-D was detected and plotted. The appropriate feed ratio was selected.

[0161] Figure 1 It can be seen that the coupling rate increases with the increase of the mass ratio of NAC to CS. There is no obvious advantage between group D (CS:NAC=1:3) and group C (CS:NAC=1:2). Considering the economy and other factors, the feed ratio of CS:NAC=1:2 is selected.

[0162] Optimization of CS-NAC reaction time

[0163] Chitosan acidification and dissolution: 100 mg of chitosan with a molecular weight of 1000 KD was weighed into a 50 mL triangular flask, 20 mL of hydrochloric acid solution with pH=5 was added, and the mixture was stirred for 1 h to swell. Four groups were prepared in parallel.

[0164] Preparation of acetylcysteine solution: 200 mg of acetylcysteine solid was weighed into a 50 mL triangular flask, 10 mL of deionized water was added, and the solution was dissolved at room temperature. Four groups were prepared in parallel.

[0165] Preparation of EDC and NHS solution: 200 mg of EDC and 100 mg of NHS were weighed and placed in a 50 mL flask, 10 mL of deionized water was added, dissolved at room temperature, and ready for use. Four groups were carried out in parallel.

[0166] Carboxyl activation: EDC and NHS solution was slowly added dropwise to the stirring NAC solution, and after the dropwise addition was completed, the reaction was stirred at room temperature for half an hour for carboxyl activation.

[0167] Coupling reaction and pH adjustment: The chitosan solution was slowly added dropwise to the stirring mixed solution of EDC, NHS and NAC, and after the dropwise addition was completed, the stirring was continued at room temperature for half an hour. Then the pH was adjusted by dropwise adding 1M sodium hydroxide solution, and the reaction solution and pH meter were observed, and after the count was stable, the dropwise addition of sodium hydroxide solution was stopped when the pH was 5.8-6.0, and the mixed solution was placed in a 4°C chromatography cabinet for stirring. The four parallel solutions were divided into A, B, C, and D groups, and reacted for 2h, 4h, 8h, and 12h, respectively.

[0168] Dialysis and freeze-drying: After stirring, the reaction solution was loaded into a dialysis bag with a molecular weight cut-off of 8000-14000 Da, and dialyzed with 3L deionized water for more than 6 times, with an interval of 6-8h each time. After dialysis, freeze-drying was carried out, and the freeze-dried product was sealed and stored in a 4°C refrigerator for standby.

[0169] The thiol coupling rate of the freeze-dried products of A-D groups was detected and plotted, and the appropriate reaction time was selected.

[0170] Figure 2 It can be seen that the coupling rate increases with the increase of time, and there is no obvious advantage between group D (reaction for 12h) and group C (reaction for 8h), considering economic factors, the reaction time of 8h is selected.

[0171] CS-NAC-DA feeding amount optimization

[0172] Thiol chitosan dissolution: 100 mg of thiol chitosan was weighed and placed in a 100 mL flask, 20 mL of deionized water solution was added, and stirred for 1 h to fully swell. Five groups were carried out in parallel.

[0173] Preparation of dopamine solution: 100 mg (group A), 150 mg (group B), 200 mg (group C), 300 mg (group D), and 400 mg (group E) of dopamine solid were weighed and placed in 50 mL flasks, respectively, 20 mL of deionized water was added, and dissolved at room temperature, ready for use.

[0174] Preparation of EDC and NHS solution: 100 mg of EDC and 50 mg of NHS (group A), 150 mg of EDC and 75 mg of NHS (group B), 200 mg of EDC and 100 mg of NHS (group C), 300 mg of EDC and 150 mg of NHS (group D), 400 mg of EDC and 200 mg of NHS (group E) were weighed into 50 mL triangular flasks, respectively, and 20 mL of deionized water was added, dissolved at room temperature, and prepared for use.

[0175] Carboxyl activation: The EDC and NHS solution was aspirated and slowly added dropwise to the dopa solution in the same group stirring, and after the dropwise addition was completed, the carboxyl activation was carried out at room temperature for half an hour.

[0176] Coupling reaction and pH adjustment: The mercapto chitosan solution was slowly added dropwise to the mixed solution of EDC, NHS and DA stirring, and after the dropwise addition was completed, the mixed solution was stirred at room temperature for half an hour. Then the pH was adjusted by dropwise adding 1M sodium hydroxide solution, and the reaction solution and pH meter were observed. When the count was stable, the dropwise addition of sodium hydroxide solution was stopped when the pH was 5.8-6.0, and the mixed solution was placed in a 4°C chromatography cabinet and stirred for 48h.

[0177] Dialysis and freeze-drying: The reaction solution was loaded into a dialysis bag with a molecular weight cut-off of 8000-14000 Da, and dialyzed with 3L deionized water for more than 6 times, with an interval of 6-8h. After dialysis, freeze-drying was carried out, and the freeze-dried product was sealed and stored in a 4°C refrigerator for standby use.

[0178] The dopa coupling rate of the freeze-dried products of groups A-E was detected and plotted, and the appropriate feed ratio was selected.

[0179] Figure 3 It can be seen that the coupling rate increases with the increase of the mass ratio of DA to CS-NAC, and there is no obvious advantage compared with group D (CS-NAC: DA = 1:3), considering the economy and other factors, the feed ratio of CS-NAC: DA = 1:3 is selected.

[0180] Optimization of CS-NAC-DA reaction time

[0181] Mercapto chitosan acidification and dissolution: 100 mg of mercapto chitosan was weighed into a 100 mL triangular flask, 20 mL of deionized water solution was added, and it was stirred for 1h to make it swell fully. Six groups were carried out in parallel.

[0182] Preparation of dopa solution: 300 mg of solid dopa was weighed into a 50 mL triangular flask, 20 mL of deionized water was added, and it was dissolved at room temperature, and prepared for use. Six groups were carried out in parallel.

[0183] Preparation of EDC and NHS solution: 300 mg of EDC and 150 mg of NHS were weighed and placed in a 50 mL flask, 20 mL of deionized water was added, dissolved at room temperature, and ready for use. Six groups were prepared in parallel.

[0184] Carboxyl activation: EDC and NHS solution was slowly added dropwise to the stirring DA solution, and after the dropwise addition was completed, the carboxyl group was activated by stirring at room temperature for half an hour.

[0185] Coupling reaction and pH adjustment: The thiol chitosan solution was slowly added dropwise to the stirring mixed solution of EDC, NHS and DA, and after the dropwise addition was completed, the mixed solution was stirred at room temperature for half an hour. Then the pH was adjusted by dropwise adding 1M sodium hydroxide solution, and the reaction solution and pH meter were observed. When the count was stable, the dropwise addition of sodium hydroxide solution was stopped, and the mixed solution was placed in a 4°C chromatography cabinet for stirring. The six parallel solutions were divided into groups A, B, C, D, E and F, and reacted for 8h, 12h, 24h, 36h, 48h and 60h, respectively.

[0186] Dialysis and freeze-drying: After stirring, the reaction solution was placed in a dialysis bag with a molecular weight cut-off of 8000-14000 Da, and dialyzed with 3L deionized water for more than 6 times, with an interval of 6-8h. After dialysis, freeze-drying was carried out, and the freeze-dried product was sealed and stored in a 4°C refrigerator for standby.

[0187] The freeze-dried products of groups A to F were detected for dopamine coupling rate and plotted, and the appropriate reaction time was selected.

[0188] Figure 4 It can be seen that the coupling rate increases with the increase of time, and there is no obvious advantage between group F (reaction for 60h) and group E (reaction for 48h). Considering the economic factors, the reaction time of 48h is selected.

[0189] Example 2, UV spectrum scanning of the adhesive layer material

[0190] A 1 mg / mL chitosan solution was prepared using hydrochloric acid with a pH of 4, a 0.1 mg / mL acetylcysteine and dopamine solution was prepared using deionized water, and a 1 mg / mL dopamine-thiolated chitosan solution was prepared. The above solutions were scanned by UV-spectrophotometer. From Figure 5 It can be seen that dopamine has ultraviolet absorption at 278 nm, acetylcysteine has no ultraviolet absorption, and the CS-NAC-DA ultraviolet spectrum scanning shows that DA is successfully coupled to the chitosan macromolecule.

[0191] Example 3, microstructure detection of the adhesive layer material

[0192] Take the appropriate amount of CS-NAC; CS-DA; CS-NAC-DA sample, respectively, prepared into 2%, 4% and 6% solution, take 2ml for freeze-drying, freeze-dried sample, scanning electron microscope (SEM) observation of the internal structure of the gel at the concentration. Figure 6 It can be seen that the CS-NAC freeze-dried sample has a neat and dense lamellar structure; the CS-DA gel freeze-dried sample has a large number of three-dimensional porous structures; the CS-NAC-DA gel freeze-dried sample has the properties of both CS-NAC and CS-DA, that is, it has both neat and dense lamellar structure and three-dimensional porous network structure. With the increase of concentration, the number of CS-NAC-DA holes increases, the pore size decreases, the lamellar density increases, and the three-dimensional network structure density increases.

[0193] Example 4, determination of the rheological properties of the adhesive layer

[0194] (1) Amplitude sweep of the rheological properties of the modified chitosan

[0195] The elastic modulus G' and viscous modulus G" of CS; CS-NAC; CS-DA; CS-NAC-DA were determined by rotary rheometer. When testing, 400 μL of solution was added to the rheometer test circle platform, and the rotary rheometer parameters were set as follows: temperature 25℃, frequency fixed at 1 rad / s, logarithmic change of strain range 0.1% to 100%, 20 points. Start the instrument, and the rheometer will automatically record the rheological data during operation. From Figure 7 It can be seen that, except for 2% CS-NAC-DA, the elastic modulus G' is less than the viscous modulus G" in the high strain interval, showing fluid properties, and the elastic modulus G' of 4% and 6% CS-NAC-DA is always greater than the viscous modulus G", proving that it always behaves as a solid gel. The linear viscoelastic region of CS-NAC-DA is about 0.1%-13% of the strain interval.

[0196] (2) Frequency sweep of the modified chitosan

[0197] The elastic modulus G' and viscous modulus G" of CS; CS-NAC; CS-DA; CS-NAC-DA were determined by rotary rheometer. When testing, 400 μL of solution was added to the rheometer test circle platform, and the rotary rheometer parameters were set as follows: temperature 25℃, strain fixed at 1%, logarithmic change of frequency range 0.1 to 100 rad / s, 20 points. Start the instrument, and the rheometer will automatically record the rheological data during operation. From Figure 8It can be seen that the 2% CS-NAC-DA elastic modulus G' and viscous modulus G" curves also intersect at about 0.12 rad / s, proving that the low concentration of CS-NAC-DA has a low crosslinking degree. The elastic modulus G' of 4% and 6% CS-NAC-DA is always greater than the viscous modulus G", proving that there is a crosslinking structure inside, which reflects the feasibility of CS-NAC-DA self-crosslinking.

[0198] (3) Time sweep of modified chitosan

[0199] The time sweep curves of CS; CS-NAC; CS-DA; CS-NAC-DA elastic modulus G' and viscous modulus G" were determined by rotary rheometer. When testing, 400 μL of solution was added to the rheometer test circle platform, and the rotary rheometer was set as follows: temperature was 25°C, strain was fixed at 1%, frequency was fixed at 1 rad / s, linear change time range was 0 to 240 s, and 20 points were taken. Start the instrument, and the rheometer will automatically record the rheological data during operation. Figure 9 It can be seen that 2% and 4% CS-NAC-DA remain good stability throughout the period, and do not appear the phenomenon of curve rising caused by oxidation as CS-DA does, indicating that CS-NAC-DA is more stable at the same concentration. The time sweep curve of 6% CS-NAC-DA appears an increase, and high concentration of CS-NAC-DA is more easily oxidized in air.

[0200] (4) Stress-strain of modified chitosan

[0201] The stress-strain curves of CS; CS-NAC; CS-DA; CS-NAC-DA were determined by rotary rheometer. When testing, 400 μL of solution was added to the rheometer test circle platform, and the rotary rheometer was set as follows: temperature was 25°C, frequency was fixed at 1 rad / s, logarithmic change strain range was 0.1% to 100%, and 20 points were taken. Start the instrument, and the rheometer will automatically record the rheological data during operation. Figure 10 It can be seen that CS-NAC-DA has high strain resistance.

[0202] Example 5, bioadhesion strength test of gel of different concentrations of CS-NAC-DA

[0203] In this experiment, the adhesion strength of the adhesive sample was tested by the international standard improved ASTM F2255-05 method. The specific operation is as follows:

[0204] First step: Prepare fresh pigskin, cut it into 2 cm wide strips, rinse it with physiological saline and wipe it dry;

[0205] Second step: Prepare samples with different concentrations, use hydrochloric acid with pH = 5 to prepare 2% CS solution, use deionized water to prepare 2%, 4% and 6% CS-NAC; CS-DA; CS-NAC-DA samples;

[0206] Third step: evenly apply 50 μL of each sample with different concentrations to the pig skin, with an application area of 2 cm x 2 cm;

[0207] Fourth step: bond the applied sample part together, use a 500g weight to press on the bonding site for 10 minutes;

[0208] Fifth step: clamp the pig skin unbonded part using the texture instrument clamp, adjust the clamp distance so that the bonded part is in an unstressed state;

[0209] Sixth step: set the texture instrument driving parameters, drive the arm to rise at a speed of 10 mm / s, stop automatically after rising 400 mm, and record the maximum tensile force during the stretching process. The ratio of the tensile force to the bonding area is the adhesion strength (unit Kpa).

[0210] Figure 11 It can be seen that the adhesion strength of 4% CS-NAC-DA and 2% CS-NAC-DA has a very significant difference (P < 0.001), and the adhesion strength of 6% CS-NAC-DA has a significant difference (P < 0.01); and the adhesion strength of 4% CS-NAC-DA and the positive control fibrin glue group has a significant difference (P < 0.0001), indicating that the synthesized modified chitosan has stronger mechanical properties than fibrin glue.

[0211] Example 6, synthesis of the base layer

[0212] The synthesis steps of the base layer are as follows:

[0213] First step: use HBSS to prepare 20 mL of 2% sodium alginate solution and 12% acrylamide solution, stir uniformly on a magnetic stirrer until the solids are completely dissolved, and remove the gas bubbles by standing;

[0214] Second step: use HBSS to prepare an appropriate amount of 2% MBAA solution, 0.375M CaSO4, and 0.27M APS;

[0215] Third step: add 72 μL of MBAA solution, 16 μL of TEMED solution and 400 μL of CaSO4 solution to the mixed solution of sodium alginate and acrylamide under high-speed stirring, and stir until the mixture is uniform;

[0216] Fourth step: quickly add 500 μL of APS to the mixed solution and stir quickly for 2 minutes;

[0217] Fifth step: Pour the mixed solution into the mold and let it crosslink overnight.

[0218] Example 7, Microstructure detection of the base layer

[0219] First step: Prepare the bulk base layer. Pour the prepared gel solution into a six-well plate and seal it for crosslinking for 48 hours. After 48 hours, remove the bulk base layer;

[0220] Second step: Slice the freeze-dried sample and observe the internal structure of the gel at the use concentration using a scanning electron microscope (SEM).

[0221] Figure 12 As can be seen, the cross-section of the base layer has a large number of holes formed by crosslinking structures, is dense and arranged in an orderly manner. The longitudinal section is similar to the cross-section, and more reflects some lamellar structure. It can be seen that the uniformity of the base layer can also be observed, and the tight and regular three-dimensional network structure of the base layer is observed, which proves the tight double crosslinking form of the gel from the microstructure.

[0222] Example 8, Rheology of the base layer

[0223] (1) Frequency sweep test of the base layer

[0224] The frequency sweep test of the base layer was determined by a rotational rheometer. When testing, a cylindrical base layer with a radius of 1 cm and a height of 0.2 cm was placed on the test circular platform of the rheometer, and the parameters of the rotational rheometer were set as follows: the temperature was 25°C, the strain was fixed at 1%, the logarithmically changed frequency range was 0.1 to 100 rad / s, and 20 points were taken. Start the instrument, and the rheometer will automatically record the rheological data during operation. Figure 13 As can be seen, the elastic modulus G' of the base layer is always much higher than the viscous modulus G", indicating that it exhibits solid properties under any condition; as the frequency increases, it still exhibits solid properties and does not break, indicating that its strength is sufficient and relatively stable.

[0225] (2) Stress-strain test of the base layer

[0226] The stress-strain curve of the base layer was determined by a rotational rheometer. When testing, a cylindrical base layer with a radius of 1 cm and a height of 0.2 cm was placed on the test circular platform of the rheometer, and the parameters of the rotational rheometer were set as follows: the temperature was 25°C, the frequency was fixed at 1 rad / s, the logarithmically changed strain range was 0.1% to 100%, and 20 points were taken. Start the instrument, and the rheometer will automatically record the rheological data during operation. From Figure 14It can be seen that the bottom almost does not receive the pressure signal before 50% strain. It shows that the internal structure of the substrate layer dissipates the strain before 50% strain, thereby proving that the substrate layer can delay and dissipate small strain without affecting the damaged tissue inside. After 50% strain, the receiver gradually receives its signal, and until 100% strain. This phenomenon shows that the substrate layer can almost reach 100% strain without disintegrating and breaking, fully proving its toughness; at the same time, the 100% strain corresponds to a pressure of 4 MPa.

[0227] Example 9, in vitro degradation of the substrate layer and the adhesive layer

[0228] First step: prepare a 0.01M PBS solution with a concentration of 1200 μg / L of lysozyme;

[0229] Second step: prepare the adhesive layer gel (i.e. 4% CS-NAC-DA gel) and the substrate layer gel;

[0230] Third step: place 1 g of the adhesive layer gel or the substrate layer gel in a 15 mL centrifuge tube, respectively, and weigh the weight of the gel + centrifuge tube; add 10 mL of lysozyme PBS solution, and replace the new lysozyme PBS solution at 6 h, 12 h, 24 h, and every other day thereafter. When replacing the solution, pour out the lysozyme PBS solution, weigh the remaining gel + centrifuge tube, and calculate the degradation rate.

[0231] From Figure 15 , Figure 16 It can be seen that the adhesive layer gel has degraded more than 50% on the 4th day, and the degradation rate gradually decreases from the 6th day, tends to be flat, and completely degrades on the 25th day. The substrate layer gel degrades to 50% on the 5th day, the degradation rate remains stable from the 6th day to the 15th day, the degradation rate gradually tends to be flat from the 15th day, and completely degrades on the 29th day. Both layers of gel have good degradability.

[0232] Example 10, in vivo degradation of the adhesive layer and the substrate layer

[0233] Experimental determination: 200 g of female SD rats were randomly divided into three groups, 12 rats in each group, to determine the degradation rate of the adhesive layer, the substrate layer, and the new biological adhesive (substrate layer loaded with adhesive layer) in rats. The specific operation is as follows:

[0234] The rats were anesthetized by intraperitoneal injection of 20% urethane, the skin on the back of the rats was cut, the degradation sample was placed subcutaneously, and the skin was sutured; the skin on the right lower limb of the rat was cut, the muscle layer was separated layer by layer until the sciatic nerve could be observed, the degradation sample was placed in the wound and above the sciatic nerve, and the skin was sutured; the skin on the right lower limb of the rat was cut, the Achilles tendon was exposed, the degradation sample was placed on the surface of the Achilles tendon, and the skin was sutured.

[0235] Group 1: This group was used to determine the degradation of the adhesive gel on the back, sciatic nerve, and Achilles tendon. 0.25g of adhesive gel was placed subcutaneously on the back of each rat, 0.05g on the sciatic nerve, and 0.05g on the Achilles tendon. A total of 12 rats were used, and 3 rats were randomly selected and sacrificed at 1 / 2 / 3 / 4 weeks post-surgery. Degradation was observed, and the remaining degraded portion was collected and its residual mass was measured.

[0236] Group 2: This group was used to determine the degradation of the basal layer in the back, sciatic nerve, and Achilles tendon. 0.3g of basal layer gel was placed subcutaneously in the back of each rat, 0.1g in the sciatic nerve, and 0.1g in the Achilles tendon. A total of 12 rats were used, and 3 rats were randomly selected and sacrificed at 1 / 2 / 3 / 4 weeks post-surgery. The degradation was observed, and the remaining degraded portion was collected and its residual mass was measured.

[0237] Group 3: Used to determine the degradation of the novel bioadhesive on the back, sciatic nerve, and Achilles tendon. The degradation mass of Group 3 was the sum of Groups 1 and 2, i.e., 0.25g of adhesive layer gel + 0.3g of basal layer gel were placed subcutaneously on the back of each rat, 0.05g of adhesive layer gel + 0.1g of basal layer gel were placed on the sciatic nerve, and 0.05g of adhesive layer gel + 0.1g of basal layer gel were placed on the Achilles tendon. A total of 12 rats were used, and 3 rats were randomly selected and sacrificed at 1 / 2 / 3 / 4 weeks after surgery. The degradation was observed, and the remaining degradation portion was removed and its residual mass was measured.

[0238] from Figure 17 As can be seen, the degradation rate of the novel bioadhesive is relatively slow. This may be because the basal layer encapsulates the adhesive layer, which greatly slows down the degradation rate of the gel. This also means that for nerve tissue, the novel bioadhesive has a longer protective effect period than a single gel or basal layer, which is in line with the conventional time for nerve tissue repair.

[0239] Example 11: Cytotoxicity of hydrogel extract

[0240] Step 1: Preparation of hydrogel DMEM extraction solution: 4% CS-NAC, 4% CS-DA, and 4% CS-NAC-DA samples were added to DMEM culture medium at a volume ratio of 1:4, and the basal layer was added at a volume ratio of 1:10. The mixture was incubated at 37℃ for 24 hours to obtain the hydrogel DMEM culture medium extract. The supernatant of the extract was used as the 100% concentration gel extract. The 100% gel extract was then serially diluted with DMEM culture medium to obtain three different concentrations: 75%, 50%, and 25%.

[0241] Second step, cell preparation: resuscitate mouse fibroblast (L929), cultivate in 37℃, relative humidity 90%, CO2 content 5% constant temperature cell incubator, change cell culture solution every two days. When the cell confluence reaches about 90%, digest and passaged with 0.25% trypsin / 0.02% EDTA, pass the cell to obtain uncontaminated and well-grown cells. Add 100 μL cell suspension of 5×104 / mL concentration in each well of 96-well culture plate, and cultivate overnight to make it adhere and grow.

[0242] Third step, sample addition:

[0243] Negative control group: add 100 μL DMEM culture solution to each well;

[0244] Experimental group: add 100 μL 25%, 50%, 75%, 100% water gel extract to each well;

[0245] Fourth step, cell culture method: place the 96-well plate in the constant temperature cell incubator for cultivation, and observe the growth of L929 cells under a microscope after 12 h;

[0246] Fifth step, MTT detection:

[0247] (1) aspirate the culture solution in each well of each group, and rinse with PBS three times;

[0248] (2) prepare 5 mg / mL MTT solution with DMEM;

[0249] (3) add 100 μL 5 mg / mL MTT solution to each well;

[0250] (4) continue to incubate in the cell incubator for 3-4 h;

[0251] (5) measure the absorbance of each well of the 96-well plate at 490 nm with an enzyme marker;

[0252] (6) calculate the relative growth rate (RGR) of each well of cells according to the measured absorbance.

[0253] Relative growth rate RGR = average absorbance of experimental group / average absorbance of negative control group × 100%.

[0254] According to the national evaluation standard of medical device biology, the toxicity degree of the material is evaluated, and the result standard is:

[0255] The toxicity degree of the material is classified as 0-1 level, which is qualified; 2 level can be comprehensively evaluated whether it is qualified in combination with cell morphology and cell growth density; and more than 3 level is unqualified. The relationship between the relative growth rate of L929 cells and the toxicity level is shown in Table 1. The RGR value and the toxicity classification of the material are shown in Table 2.

[0256] Table 1 L929 cell relative proliferation rate and toxicity reaction level control table

[0257]

[0258]

[0259] Table 2 L929 cell relative proliferation rate and toxicity reaction level control table

[0260]

[0261] Figure 18 As can be seen from Table 2, CS-NAC-DA and the base layer are both 0 or 1 level of toxicity, indicating that the material has good biocompatibility.

[0262] Example 12, rat sciatic nerve modeling

[0263] First step: anesthetize the rats by intraperitoneal injection of urethane solution (20%, w / v), and the anesthetic dose is 5 mL / kg;

[0264] Second step: after the rat is anesthetized, fix the rat prone, trim the right back and right hind leg fur of the rat appropriately, and then apply a small amount of depilatory cream to the surgical site to remove all the fur, so as to avoid the fur falling into the field of view during the subsequent operation, or causing postoperative infection. Use gauze to clean the rat's back with 75% alcohol and disinfect it;

[0265] Third step: use a sterilized surgical scissors to cut the rat's outer skin along the line of the rat's right hind leg muscle, and then cut the inner skin. Use a sterilized needle holder to bluntly separate the rat's hind leg fascia layer and muscle layer, and expose the sciatic nerve.

[0266] Fourth step: use a sterilized microscissors to completely cut the sciatic nerve;

[0267] Fifth step: according to the different experimental groups, different treatments are performed on the rats after the modeling is completed, and then the sciatic nerve is returned to its original position;

[0268] Sixth step: suture the rat's outer skin with surgical sutures. According to the experimental scheme, observe the state of the rat regularly and record the relevant data.

[0269] Thirty-six SD rats of the same batch were randomly and evenly divided into 6 groups, with 6 rats in each group. The grouping settings are shown in Table 3. Three rats were randomly selected from each group every week for rat walking stride determination; five rats were randomly selected from each group every week for grip strength determination. At the sixth week, the rats were sacrificed, and three rats were randomly selected from each group for nerve pull-off force determination experiment; three rats were selected for IL-6 content detection of the sciatic nerve tissue at the surgical site.

[0270] Table 3 Rat grouping for sciatic nerve injury repair experiment

[0271]

[0272] Example 13, Evaluation of the repair effect of the rat sciatic nerve transection experiment

[0273] (1) Fix the SD rats, expose their right hind paws, and press their right hind paws on red ink for 20 seconds to make them have enough red pigment. Release the rats and guide them to walk normally on the white paper. Collect the white paper with the marks and measure the distance between the rat paw prints. Randomly select three rats from each group for this experiment.

[0274] The experimental results are shown in Table 1. Figure 19 The average step distance of the control group is always higher than that of the four repair groups (C-F), and the significant difference gradually decreases, and the gap gradually decreases with the recovery process. At the same time, the average step distance of the four repair groups (C-F) is higher than that of the transection group, indicating that the four methods have certain repair effect.

[0275] Fibrin glue is a medical material that has been marketed and can be used for nerve repair. In this experiment, it was selected as a positive control. Single factor ANOVA (ANOVA) found that there was no significant difference between the two groups. Since the data of the experimental group is always higher than that of the positive control group, the method used in the experimental group is obviously not inferior to the method used in the positive control group. Therefore, we conducted a superiority test on the positive control group and the experimental group. After calculation, the t value from the first week to the sixth week is greater than the T value corresponding to the 95% confidence interval. It can be concluded that the step distance recovery effect of the method used in the experimental group is better than that of the fibrin glue + suture one needle method.

[0276] The surgical group used the most commonly used epineurium suture method in current clinical surgery, which can butt the transected nerve together as much as possible without damaging the endoneurium. The difference between the surgical group and the experimental group is very small, and the ANOVA test shows that there is no significant difference between the two methods. A superiority test was conducted on the surgical group and the experimental group. After calculation, the t value from the first week to the sixth week is less than the T value corresponding to the 95% confidence interval. It cannot be concluded that the step distance recovery effect of the method used in the experimental group is better than that of the surgical method. Next, a non-inferiority test was conducted on the surgical group and the experimental group. After calculation, the t value of the first week is less than the T value corresponding to the 95% confidence interval, and the t value from the second week to the sixth week is greater than the T value corresponding to the 95% confidence interval. It can be concluded that the step distance recovery effect of the method used in the experimental group is not inferior to that of the surgical method.

[0277] (2) Fix the SD rats, expose their right hind paws, and make them grip the grip tester with their right hind paws. Adjust the position of the mouse so that the mouse grips the tester without conscious and external force and pulls it back. The grip tester can record the maximum grip strength of the right hind limb of the rat. Randomly select five rats from each group for this experiment.

[0278] The experimental results are shown in Table 1. Figure 20 From the fourth week, the average grip strength recovery of the experimental group I was significantly better than that of the positive control group. The positive control group and the experimental group I were subjected to efficacy test, and it was concluded that the method used in the experimental group I was better than the fibrin glue + suture needle method in long-term recovery of grip strength.

[0279] The surgical group and the experimental group I were subjected to efficacy test and non-inferiority test, and it was concluded that the method used in the experimental group I was not inferior to the surgical method in recovery of grip strength.

[0280] (3) After six weeks of modeling, the rats were killed by intraperitoneal injection of excess urethane, and the surgical site sciatic nerve of the rats was dissected. The tensile strength of the repaired nerve was tested using a material property instrument. The surgical site sciatic nerve was fixed up and down using a clamp, and the clamp distance was adjusted before the test to make the nerve in a stress-free state. The driving parameters of the material property instrument were set, and the driving arm was raised at a speed of 10 mm / s and automatically stopped after rising 400 mm. The material property instrument automatically recorded the force generated when the surgical site sciatic nerve was disconnected, i.e. the nerve tensile strength. Three rats were randomly selected from each group for the test.

[0281] The experimental results are shown in Table 1. Figure 21As shown in the figure, the sciatic nerve rupture force in the control group rats reached the maximum value measured by the instrument (5N), indicating that the normal sciatic nerve can withstand a tensile force of 5N. In the transected group rats, only slight reconnection was observed after six weeks, and one rat showed no reconnection between the nerve ends, indicating that nerve repair is very poor without any treatment after nerve rupture. All repair groups showed significant differences compared to the transected group, demonstrating that the nerve reconnection in each repair group was significantly stronger than that in the transected group. The average nerve rupture force in the positive control group was stronger than that in the transected group, but still significantly weaker than that in the normal control group (P<0.001), indicating that the fibrin glue repair method has only a weak repair effect and differs significantly from the normal level. Surgical repair is currently the most effective clinical method for repair; the nerve rupture force in the surgical group rats reached 5N, which was on par with the control group. Similar to the surgical group and the control group, all rats in Experimental Group 1 achieved a nerve rupture force of 5N, indicating that within the measurement range of this instrument, the repair effect of the method used in Experimental Group 1 was no less than that of the surgical method. Furthermore, both methods showed significant differences compared to the positive control group (P<0.001), suggesting that these two methods are superior to the fibrin glue bonding method. The average nerve rupture force in Experimental Group 2 was higher than that in the transected group and the positive control group, but there was no significant difference compared to the positive control group, indicating that the repair effect of this method was slightly better than that of fibrin glue. The average rupture force was lower than that in the surgical group and Experimental Group 1, and there was a significant difference compared to them (P<0.01), indicating that at six weeks, the repair effect of this method was weaker than that of the surgical method and the synergistic method.

[0282] Example 14: Measurement of IL-6 cytokine levels

[0283] Six weeks after modeling, rats were euthanized by intraperitoneal injection of an overdose of urethane. The sciatic nerve at the surgical site was dissected and ground using a tissue homogenizer at -10℃. The homogenization conditions were: 70 Hz, 45 s running time, 15 s pause, and 30 runs. After homogenization, the tissue was centrifuged at 3000 rpm for 10 min at 4℃. The supernatant was collected to obtain the tissue homogenate of the damaged and repaired nerve tissue. The content of the inflammatory factor IL-6 in the tissue homogenate of the repaired nerve tissue of each group was measured using a rat IL-6 ELISA kit. Three rats were randomly selected from each group for this experiment. The measurement procedure is as follows:

[0284] First, remove the reagent kit from the 4°C refrigerator 30 minutes before the experiment, allow it to return to room temperature, wash the plate 3 times, and shake it thoroughly on filter paper;

[0285] The second step is to add 100 μL of a series of gradient standards and the serum sample to be tested into the reaction wells, seal the plate, and incubate it in a 37°C incubator for 90 min.

[0286] Third step, wash the plate 4 times and dry on filter paper, add 100 μL biotinylated antibody working solution to each reaction well, seal the plate, and incubate in a 37°C incubator for 60 min;

[0287] Fourth step, wash the plate 4 times and dry on filter paper, add 100 μL enzyme conjugate working solution to each reaction well, seal the plate, and incubate in a 37°C incubator for 30 min;

[0288] Fifth step, wash the plate 5 times and dry on filter paper, add 100 μL color developing substrate to each reaction well, seal the plate, and incubate in a 37°C incubator for 15 min in the dark;

[0289] Sixth step, add 50 μL stop solution to each reaction well, and measure the OD value of each reaction well at 450 nm wavelength within 5 min using an enzyme label instrument.

[0290] The experimental results are shown in Table 1. Figure 22 The value of the positive control group is less than that of the operation group and the experimental group one, indicating that the inflammatory reaction caused by the fibrin glue is smaller; the value of the operation group is slightly higher than that of the experimental group one, indicating that the foreign body sensation and inflammatory reaction caused by the suture thread are higher than those of the new biological adhesive, indicating that the method used in the experimental group one is not inferior to the operation method in terms of biocompatibility; the value of the experimental group two is lower than that of the experimental group one, indicating that the suture thread does indeed have an adverse effect on the body.

[0291] Example 15, rat tendon modeling

[0292] First step: anesthetize the rats by intraperitoneal injection of urethane solution (20%, w / v), and the anesthetic dose is 5 mL / kg;

[0293] Second step: after the rat anesthesia is completed, fix the rat in a prone position, trim the rat's right hind leg fur appropriately, and apply a small amount of depilatory cream to completely remove the fur at the operation site. Use gauze dipped in 75% alcohol to clean the rat's leg and disinfect it;

[0294] Third step: use a sterilized surgical scissors to cut the rat's leg skin along the line, and then cut the inner skin to expose the Achilles tendon.

[0295] Fourth step: completely cut the Achilles tendon with a sterilized microscissors;

[0296] Fifth step: according to the different experimental groups, different treatments are performed on the rats after the modeling is completed, and after the treatment is completed, the Achilles tendon is returned to its original position;

[0297] Sixth step: suture the rat's skin with surgical suture thread. According to the experimental scheme, observe the state of the rat regularly and record the relevant data.

[0298] 30 SD rats of the same batch were randomly divided into 5 groups, 6 rats in each group. The group setting is shown in Table 4. 3 rats were randomly selected from each group every week for the determination of the rat walking stride; 3 rats were randomly selected from each group every week for the determination of the grip strength. The Achilles tendon breaking force determination experiment and the IL-6 content of the Achilles tendon tissue at the surgical site were performed on 3 rats randomly selected from each group at the second and fourth weeks.

[0299] Table 4 Rat grouping for Achilles tendon injury repair experiment

[0300]

[0301] Example 16, Evaluation of the repair effect of rat Achilles tendon transection experiment

[0302] (1) Stride determination experiment: the same as the experimental procedure of Example 13.

[0303] The experimental results are shown in the figure. Within four weeks, the average stride of the control group was higher than that of the four repair groups (C~F), and the significant difference between them gradually decreased or even disappeared, indicating that the model group was still different from the normal rats within four weeks, but the repair level had approached that of the normal rats. The average stride of the four repair groups (C~F) was higher than that of the transection group, and the significant difference between them gradually increased, indicating that the repair rate of the four groups was higher than that of the transection group.

[0304] Surgical method is a common method for repairing Achilles tendon in current clinical practice, and is one of the methods with better repair effect. The first surgical group adopts the conventional suture needle method (hereinafter referred to as surgical method one), which is mainly used as a control for the first experimental group. The second surgical group adopts the suture four-needle method (hereinafter referred to as surgical method two), which is mainly used as a positive control.

[0305] Figure 23 It can be seen that there is no significant difference between the second surgical group and the first surgical group within four weeks by ANOVA test. Therefore, the superiority test was performed on the second surgical group and the first experimental group. The critical value δ0 is 0, and the confidence interval is 95%. After calculation, the t value from the first week to the fourth week is less than the T value corresponding to the 95% confidence interval, so it cannot be concluded that the stride recovery effect of the method used in the first experimental group is superior to that of the surgical method. Next, the non-inferiority test was performed on the surgical group and the first experimental group. The difference between the average stride of the second surgical group and the transection group after four weeks is taken as the effect difference when the positive control drug is compared with the placebo, the critical value δ0 is taken as 1 / 2 of the effect difference, about 1.192 cm, and the confidence interval is 95%. After calculation, the t value of the first week and the second week is less than the T value corresponding to the 95% confidence interval, and the t value of the third week and the fourth week is greater than the T value corresponding to the 95% confidence interval, so it can be concluded that the long-term stride recovery effect of the method used in the first experimental group is not inferior to that of the method used in the second surgical group.

[0306] (2) Grip strength test: same as the procedure of Example 13.

[0307] Figure 24 It can be seen that within four weeks, there was no significant difference between the operation group two and the experimental group one by ANOVA test. Therefore, the superiority test was performed on the operation group and the experimental group one. The critical value δ0 was taken as 0, and the confidence interval was 95%. After calculation, the t value from the first week to the fourth week was less than the T value corresponding to the 95% confidence interval, and it could not be concluded that the grip strength recovery effect of the method used in the experimental group one was better than that of the operation method. Next, we performed a non-inferiority test on the operation group and the experimental group one. The difference between the mean grip strength of the operation group two and the transection group after four weeks was taken as the effect difference when the positive control drug was compared with the placebo, the critical value δ0 was taken as 1 / 2 of the effect difference, about 73.4N, and the confidence interval was 95%. After calculation, except for the second week, the t value from the first week to the fourth week was greater than the T value corresponding to the 95% confidence interval, and it could be concluded that the grip strength recovery effect of the method used in the experimental group one was not inferior to that of the operation group two.

[0308] (3) After two / four weeks of modeling, the rats were sacrificed by intraperitoneal injection of excess urethane, and the Achilles tendons at the surgical site were dissected. The tensile strength of the repaired Achilles tendon was tested using a material property instrument. The surgical site Achilles tendon was fixed up and down using a clamp, and the clamp distance was adjusted before the test to make the Achilles tendon in the unstressed state. The driving parameters of the material property instrument were set, and the driving arm was raised at a speed of 10 mm / s and automatically stopped after rising 400 mm. The material property instrument automatically recorded the force generated when the surgical site Achilles tendon was disconnected, i.e. the tensile strength of the Achilles tendon. Three rats were randomly selected from each group for this experiment.

[0309] The Achilles tendon belongs to muscle tissue, which will repair and heal itself after injury. The speed of healing is the repair rate, and the degree of healing is the repair effect. In this experiment, the required force to break the Achilles tendon was measured to determine the healing degree of the injured Achilles tendon, thereby quantitatively representing the repair effect. We measured the tensile strength of the Achilles tendon at the second and fourth weeks to determine the repair progress of the Achilles tendon.

[0310] Figure 25It can be seen that the control group can bear 5N force at the second week. The cross-section group cannot bear its own weight because the tendon has not healed or the healing part is not enough to bear the weight, so the breaking force cannot be measured, which is shown as 0N. The remaining four groups have a certain value, which shows that the four methods can promote tendon repair. The average breaking force of the tendon of the surgical group one, the experimental group one, the experimental group two and the surgical group two is less than that of the surgical group two, which shows that the repair effect of the second surgical method is the best at the second week. The surgical group one and the experimental group two have significant difference compared with the surgical group two (p<0.01), but the experimental group one has no significant difference compared with the surgical group two, and the numerical difference is very small, which shows that the repair effect of the experimental group one is only slightly lower than that of the surgical group two, and there is no obvious difference. The surgical group one and the experimental group two have significant difference compared with the experimental group one, which shows that the repair effect of the synergistic method of the new biological adhesive and the single suture is better than that of the two single methods at the second week.

[0311] Figure 26 It can be seen that the control group, the surgical group two and the experimental group one can bear 5N force at the fourth week, which shows that the repair degree of the surgical group two and the experimental group one using the method is close to that of the normal rat in the determination range of the instrument at four weeks, and the repair effect of the experimental group one is not inferior to that of the surgical group two. The surgical group one has significant difference compared with the surgical group two (p<0.05), which shows that the surgical method one still has a gap compared with the surgical method two; because the data of the surgical group two and the experimental group one are the same, it also shows that the repair effect of the surgical method one is weaker than that of the synergistic method, which proves the functionality of the new biological adhesive. The experimental group two has no significant difference compared with the experimental group one, but the numerical value is still lower than that of the experimental group one, which shows that the recovery effect of the experimental group two is close to that of the experimental group one at the fourth week, but it is still weaker than that of the experimental group one. The experimental group two has no significant difference compared with the surgical group one, and the numerical value is higher than that of the surgical group one, which shows that the effect of the new biological adhesive bonding method on the reconnection of the tendon at the fourth week exceeds that of the surgical method one in data.

[0312] Example 17, IL-6 cytokine content determination

[0313] After two / four weeks of modeling, the rats were killed by intraperitoneal injection of excess urethane, and the rat surgical site tendons were dissected out. A tissue grinder was used for grinding at-10℃, and the grinding conditions were as follows: running frequency 70HZ, running time 45s, pause time 15s, and running number 50 times. After grinding, a centrifuge was used for centrifugation at 3000rpm at 4℃ for 10min, and the supernatant was taken to obtain the tissue grinding liquid of the rat injured and repaired tendon tissue. The content of inflammatory factor IL-6 in the tissue grinding liquid of the rat repaired tendon tissue of each group was determined by using a rat IL-6 ELISA kit. Three rats were randomly selected from each group for the experiment. The determination steps were the same as those of Example 14.

[0314] Figure 27It can be seen that in the second week, the first surgical group and the second experimental group have significant differences compared with the first experimental group, which shows that the synergistic repair method of the new biological adhesive and one needle suture causes more inflammatory reactions than the two single methods.

[0315] Figure 28 It can be seen that in the fourth week, the second surgical group has significant differences compared with the first experimental group, especially in the second week, the IL-6 content of the first experimental group is slightly higher than that of the second surgical group, and in the fourth week, the IL-6 content of the first experimental group is lower than that of the second surgical group, which has significant differences, indicating that the inflammatory reaction caused by four needle sutures is more persistent, and the synergistic method of the new biological adhesive and one needle suture is superior to the second surgical method in biocompatibility.

[0316] The second experimental group has significant differences compared with the first experimental group, which shows that the suture in the synergistic method will cause more persistent inflammatory reactions.

[0317] Example 18, biological adhesive

[0318] (1) Preparation of acetylcysteine-coupled thiolated chitosan (CS-NAC):

[0319] Chitosan (CS) acidification and dissolution: 500 mg of chitosan with a molecular weight of 1000 KDa was placed in a 100 mL triangular flask, 30 mL of hydrochloric acid solution with pH = 5 was added, and it was stirred for 2 h to make it fully swollen;

[0320] Preparation of acetylcysteine (NAC) solution: 1000 mg of solid acetylcysteine was placed in a 50 mL triangular flask, 20 mL of deionized water was added, and it was dissolved at room temperature for standby;

[0321] Preparation of EDC and NHS solution: 1000 mg of EDC and 500 mg of NHS were placed in a 50 mL triangular flask, 10 mL of deionized water was added, and it was dissolved at room temperature for standby;

[0322] Carboxyl activation: slowly add the EDC and NHS solution drop by drop to the stirring NAC solution, after the addition is completed, stir at room temperature for half an hour for carboxyl activation;

[0323] Coupling reaction and pH adjustment: slowly add the chitosan solution drop by drop to the stirring mixed solution of EDC, NHS and NAC, after the addition is completed, continue to stir at room temperature for half an hour; then adjust the pH by dropping 1M sodium hydroxide solution, and observe the reaction solution and pH meter count, stop dropping sodium hydroxide solution after the count is stable to 5.8-6.0, and place the mixed solution in a 4℃ chromatography cabinet and stir for 8 h;

[0324] Dialysis and freeze-drying: After stirring, the reaction solution was loaded into a dialysis bag with a molecular weight cut-off of 8000-14000 Da, dialyzed with 3 L of deionized water for more than 6 times, with an interval of 6-8 h between each dialysis; after dialysis, freeze-drying was performed, and CS-NAC was obtained; the freeze-dried product was sealed and stored in a 4°C refrigerator for standby;

[0325] (2) Preparation of thiolated chitosan coupled with dopamine and acetylcysteine (CS-NAC-DA):

[0326] CS-NAC dissolution: 500 mg of CS-NAC was weighed into a 200 mL triangular flask, 50 mL of deionized water solution was added, and stirring was performed for 1 h to fully swell;

[0327] Preparation of dopamine (DA) solution: 1500 mg of DA solid was weighed into a 50 mL triangular flask, 50 mL of deionized water was added, and it was dissolved at room temperature for standby;

[0328] Preparation of EDC and NHS solution: 1500 mg of EDC and 750 mg of NHS were weighed into a 50 mL triangular flask, 20 mL of deionized water was added, and it was dissolved at room temperature for standby;

[0329] Carboxyl activation: The EDC and NHS solution was sucked out and slowly added dropwise to the stirring DA solution, and after the addition was completed, carboxyl activation was performed by stirring at room temperature for half an hour;

[0330] Coupling reaction and pH adjustment: The CS-NAC solution was slowly added dropwise to the mixed solution of EDC, NHS and DA under stirring, and after the addition was completed, stirring was continued at room temperature for half an hour; after the addition was completed, stirring was continued at room temperature for half an hour; then the pH was adjusted by dropwise adding 1 M sodium hydroxide solution, and the reaction solution and pH meter were observed; after the count was stable, the dropwise addition of sodium hydroxide solution was stopped when the pH reached 5.8-6.0, and the mixed solution was placed in a 4°C chromatography cabinet for stirring for 48 h;

[0331] Dialysis and freeze-drying: The reaction solution was loaded into a dialysis bag with a molecular weight cut-off of 8000-14000 Da, dialyzed with 3 L of deionized water for more than 6 times, with an interval of 6-8 h between each dialysis; after dialysis, freeze-drying was performed, and CS-NAC-DA was obtained; the freeze-dried product was sealed and stored in a 4°C refrigerator for standby;

[0332] (3) Preparation of the base layer:

[0333] First step: 2% sodium alginate solution and 12% acrylamide solution 20 mL were prepared using HBSS buffer, stirred uniformly on a magnetic stirrer until the solids were completely dissolved, and the bubbles were removed by standing;

[0334] Second step: prepare 2% methylene bisacrylamide (MBAA) solution, 0.375M calcium sulfate (CaSO4) solution, 0.27M ammonium persulfate (APS) solution using HBSS buffer solution;

[0335] Third step: add 72μL MBAA solution, 16μL tetramethyl ethylenediamine (TEMED) and 400μL CaSO4 solution into the mixed solution of sodium alginate and acrylamide under high-speed stirring, and stir until mixed evenly;

[0336] Fourth step: quickly add 500μL APS solution into the mixed solution, and stir quickly for 2 minutes;

[0337] Fifth step: pour the mixed solution into the mold, and place it overnight to crosslink, thereby obtaining the base layer;

[0338] (4) Preparation of the novel biological adhesive:

[0339] Prepare 4% CS-NAC-DA gel, use a 1mL syringe to take an appropriate amount of the gel, and slowly drop it onto the surface of the base layer, and spread it evenly, and cut it into appropriate size according to the need, thereby obtaining the novel biological adhesive.

[0340] Example 19, biological adhesive

[0341] (1) Preparation of acetylcysteine-coupled mercaptized chitosan (CS-NAC):

[0342] Chitosan (CS) acidification and dissolution: weigh 250mg of chitosan into a triangular flask, add hydrochloric acid solution, and stir to swell completely;

[0343] Prepare acetylcysteine (NAC) solution: weigh 500mg of solid acetylcysteine into a triangular flask, add deionized water, and dissolve at room temperature for standby;

[0344] Prepare EDC and NHS solution: weigh 500mg of EDC and 250mg of NHS into a triangular flask, add deionized water, and dissolve at room temperature for standby;

[0345] Carboxyl activation: slowly add the EDC and NHS solution dropwise into the stirring NAC solution, and after the addition is completed, stir at room temperature to carry out carboxyl activation;

[0346] Coupling reaction and pH adjustment: slowly add the chitosan solution dropwise into the stirring mixed solution of EDC, NHS and NAC, after the addition is completed, continue to stir at room temperature, and then adjust the pH to 5.8 by dropwise adding sodium hydroxide solution, and place the mixed solution in a chromatography cabinet for stirring;

[0347] Dialysis and freeze-drying: the reaction solution was loaded into dialysis bag, dialyzed with deionized water, and freeze-dried after dialysis, to obtain CS-NAC.

[0348] (2) Preparation of thiolated chitosan coupled with dopamine and acetylcysteine (CS-NAC-DA):

[0349] Dissolution of CS-NAC: 250 mg of CS-NAC was weighed into a triangular flask and deionized water was added to make it swell completely;

[0350] Preparation of dopamine (DA) solution: 750 mg of DA solid was weighed into a triangular flask and deionized water was added to dissolve at room temperature;

[0351] Preparation of EDC and NHS solution: 750 mg of EDC and 300 mg of NHS were weighed into a triangular flask and deionized water was added to dissolve at room temperature;

[0352] Carboxyl activation: the EDC and NHS solution was sucked out and slowly added dropwise into the stirring DA solution, and after the addition was completed, the carboxyl activation was carried out by stirring at room temperature;

[0353] Coupling reaction and pH adjustment: the CS-NAC solution was slowly added dropwise into the stirring mixed solution of EDC, NHS and DA, and after the addition was completed, stirring was continued at room temperature. After the addition of sodium hydroxide solution was completed, the pH was adjusted to 5.8, and the mixed solution was placed in a chromatography cabinet for stirring;

[0354] Dialysis and freeze-drying: the reaction solution was loaded into dialysis bag, dialyzed with deionized water, and freeze-dried after dialysis, to obtain CS-NAC-DA.

[0355] (3) Preparation of the base layer:

[0356] First step: 1% sodium alginate solution and 6% acrylamide solution were prepared using HBSS buffer (Hank's Balanced Salt Solution) and stirred on a magnetic stirrer until the solids were completely dissolved. After standing for a while, the bubbles were removed;

[0357] Second step: 1% methylene bisacrylamide (MBAA) solution, 0.1M calcium sulfate (CaSO4) solution, and 0.1M ammonium persulfate (APS) solution were prepared using HBSS buffer;

[0358] Third step: 36 μL of MBAA solution, 8 μL of tetramethyl ethylenediamine (TEMED), and 200 μL of CaSO4 solution were added dropwise into the stirring sodium alginate and acrylamide mixed solution, and stirring was continued until the mixture was homogeneous;

[0359] Fourth step: 250 μL of APS solution was quickly added into the mixed solution and stirred rapidly;

[0360] Fifth step: The mixed solution was poured into the mold and left overnight to crosslink, thus obtaining the base layer.

[0361] (4) Preparation of the novel bioadhesive:

[0362] A 6% CS-NAC-DA gel was prepared, and an appropriate amount of the gel was slowly added onto the surface of the base layer using a syringe and spread evenly. The novel bioadhesive was cut into appropriate sizes as needed.

Claims

1. A bio-adhesive that promotes the repair of peripheral nerve injuries, characterized in that, Prepared by the following method: (1) Preparation of acetylcysteine-coupled thiolated chitosan CS-NAC: (1.1) Chitosan CS acidification and dissolution: Weigh chitosan and place it in an Erlenmeyer flask, add hydrochloric acid solution, stir to make it fully swell and dissolve, and obtain chitosan solution; (1.2) Preparation of acetylcysteine ​​NAC solution: Weigh the solid acetylcysteine ​​and place it in an Erlenmeyer flask, add deionized water, dissolve at room temperature, and set aside. (1.3) Preparation of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC and N-hydroxysulfonic acid succinimide NHS solution: Weigh EDC and NHS into an Erlenmeyer flask, add deionized water, dissolve at room temperature, and set aside. (1.4) Carboxyl activation: EDC and NHS solution were slowly added dropwise to NAC solution under stirring. After the addition was completed, the reaction was stirred at room temperature to activate the carboxyl group. (1.5) Coupling reaction and pH adjustment: The chitosan solution was slowly added dropwise to the mixed solution of EDC, NHS and NAC while stirring. After the addition was completed, stirring was continued at room temperature and the pH value was adjusted. (1.6) Dialysis and freeze-drying: After stirring, the reaction solution was placed in a dialysis bag and dialyzed with deionized water. After dialysis, the solution was freeze-dried to obtain CS-NAC. (2) Preparation of thiolated chitosan CS-NAC-DA coupled with dopa and acetylcysteine: (2.1) Dissolving CS-NAC: Weigh CS-NAC and place it in an Erlenmeyer flask, add deionized water solution, and stir to make it fully swollen; (2.2) Preparation of dopa DA solution: Weigh DA solid and place it in an Erlenmeyer flask, add deionized water, dissolve at room temperature, and set aside. (2.3) Preparation of EDC and NHS solution: Weigh EDC and NHS into an Erlenmeyer flask, add deionized water, dissolve at room temperature, and set aside. (2.4) Carboxyl activation: The EDC and NHS solutions were removed and slowly added dropwise to the DA solution under stirring. After the addition was completed, the reaction was stirred at room temperature to activate the carboxyl groups. (2.5) Coupling reaction and pH adjustment: The CS-NAC solution was slowly added dropwise to the mixed solution of EDC, NHS and DA under stirring. After the addition was completed, the mixture was stirred at room temperature and the pH was adjusted. The mixed solution was then placed in the chromatography cabinet and stirred. (2.6) Dialysis and freeze-drying: The reaction solution was placed in a dialysis bag and dialyzed with deionized water. After dialysis, the solution was freeze-dried to obtain CS-NAC-DA. (3) Preparation of the base layer: (3.1) Use HBSS buffer to prepare sodium alginate solution and acrylamide solution, stir evenly on a magnetic stirrer until the solid is completely dissolved, and let stand to remove air bubbles; (3.2) Prepare appropriate amounts of methylenebisacrylamide (MBAA) solution, calcium sulfate (CaSO4) solution, and ammonium persulfate (APS) solution using HBSS buffer. (3.3) Add the MBAA solution, tetramethylethylenediamine (TEMED) and CaSO4 solution dropwise to the sodium alginate and acrylamide mixture under high-speed stirring, and stir until the mixture is homogeneous; (3.4) Quickly add the APS solution to the mixed solution and stir rapidly; (3.5) Pour the mixed solution into the mold and let it stand overnight to allow it to crosslink, thus obtaining the base layer; (4) Preparation of novel bioadhesives: A suitable amount of CS-NAC-DA gel is drawn up using a syringe and slowly spread on the surface of the basal layer to obtain a novel bioadhesive.

2. The bio-adhesive according to claim 1, characterized in that, Prepared by the following method: (1) Preparation of acetylcysteine-coupled thiolated chitosan CS-NAC: Chitosan CS acidification and dissolution: Weigh 250-1000 mg of chitosan and place it in an Erlenmeyer flask. Add hydrochloric acid solution and stir to allow it to swell and dissolve fully, thus obtaining a chitosan solution. Preparation of acetylcysteine ​​(NAC) solution: Weigh 500-2000 mg of solid acetylcysteine ​​into an Erlenmeyer flask, add deionized water, dissolve at room temperature, and set aside. Preparation of EDC and NHS solution: Weigh 500-2000 mg of EDC and 250-1000 mg of NHS into an Erlenmeyer flask, add deionized water, dissolve at room temperature, and set aside. Carboxyl activation: EDC and NHS solution were slowly added dropwise to NAC solution while stirring. After the addition was complete, the reaction was stirred at room temperature to activate the carboxyl groups. Coupling reaction and pH adjustment: The chitosan solution was slowly added dropwise to the mixed solution of EDC, NHS and NAC while stirring. After the addition was complete, stirring was continued at room temperature. Then, the pH was adjusted to 5.8-6.0 by adding sodium hydroxide solution dropwise. The mixed solution was then placed in the chromatography cabinet and stirred. Dialysis and freeze-drying: After stirring, the reaction solution is placed in a dialysis bag, dialyzed with deionized water, and then freeze-dried after dialysis to obtain CS-NAC.

3. The bio-adhesive according to claim 1, characterized in that, (2) Preparation of thiolated chitosan CS-NAC-DA coupled with dopa and acetylcysteine: CS-NAC dissolution: Weigh 250-1000 mg of CS-NAC into an Erlenmeyer flask, add deionized water solution, and stir to allow it to swell fully; Preparation of DOPA (Dopamine) solution: Weigh 750-3000 mg of solid DA into an Erlenmeyer flask, add deionized water, dissolve at room temperature, and set aside. Preparation of EDC and NHS solution: Weigh 750-3000 mg of EDC and 300-1500 mg of NHS into an Erlenmeyer flask, add deionized water, dissolve at room temperature, and set aside. Carboxyl activation: The EDC and NHS solutions were removed and slowly added dropwise to the DA solution under stirring. After the addition was complete, the reaction was stirred at room temperature to activate the carboxyl groups. Coupling reaction and pH adjustment: Slowly add CS-NAC solution dropwise to a mixed solution of EDC, NHS and DA under stirring. After the addition is complete, stir at room temperature. Continue stirring at room temperature until the pH is adjusted to 5.8-6.0 by adding sodium hydroxide solution. Then stop adding sodium hydroxide solution and place the mixed solution in the chromatography cabinet for stirring. Dialysis and freeze-drying: The reaction solution is placed in a dialysis bag and dialyzed with deionized water. After dialysis, the solution is freeze-dried to obtain CS-NAC-DA.

4. The bio-adhesive according to claim 1, characterized in that, (3) Preparation of the base layer: Step 1: Prepare a 1-4% sodium alginate solution and a 6-24% acrylamide solution using HBSS buffer. Stir on a magnetic stirrer until the solids are completely dissolved, and let stand to remove air bubbles. Step 2: Prepare an appropriate amount of 1-3% methylenebisacrylamide (MBAA) solution, 0.1-0.75M calcium sulfate (CaSO4) solution, and 0.1-0.5M ammonium persulfate (APS) solution using HBSS buffer. Step 3: Add 36-140 μL MBAA solution, 8-32 μL tetramethylethylenediamine (TEMED) and 200-400 μL CaSO4 solution dropwise to the sodium alginate and acrylamide mixed solution under high-speed stirring, and stir until the mixture is homogeneous. Step 4: Quickly add 250-1000 μL of APS solution to the mixed solution and stir rapidly; Step 5: Pour the mixed solution into the mold and let it stand overnight to allow it to crosslink, thus obtaining the base layer.

5. The bio-adhesive according to claim 1, characterized in that, (4) Preparation of novel bioadhesives: Prepare 2-8% CS-NAC-DA gel, use a syringe to slowly drip an appropriate amount onto the surface of the basal layer, and spread it evenly. Cut it into appropriate sizes as needed to obtain a novel bioadhesive.

6. The bio-adhesive according to claim 1, characterized in that, Includes the following steps: (1) Preparation of acetylcysteine-coupled thiolated chitosan CS-NAC: Chitosan CS acidification and dissolution: Weigh 500 mg of chitosan with a molecular weight of 1000 kDa and place it in a 100 mL Erlenmeyer flask. Add 30 mL of hydrochloric acid solution with pH = 5 and stir for 2 h to allow it to swell fully. Preparation of acetylcysteine ​​(NAC) solution: Weigh 1000 mg of solid acetylcysteine ​​into a 50 mL Erlenmeyer flask, add 20 mL of deionized water, dissolve at room temperature, and set aside. Preparation of EDC and NHS solution: Weigh 1000 mg of EDC and 500 mg of NHS into a 50 mL Erlenmeyer flask, add 10 mL of deionized water, dissolve at room temperature, and set aside. Carboxyl activation: EDC and NHS solution were slowly added dropwise to NAC solution under stirring. After the addition was complete, the mixture was stirred at room temperature for half an hour to activate the carboxyl groups. Coupling reaction and pH adjustment: Chitosan solution was slowly added dropwise to a mixed solution of EDC, NHS and NAC under stirring. After the addition was complete, stirring was continued at room temperature for half an hour. Then, the pH was adjusted by adding 1M sodium hydroxide solution dropwise, and the reaction solution and pH meter count were observed. After the count stabilized at 5.8-6.0, the addition of sodium hydroxide solution was stopped, and the mixed solution was placed in a chromatography cabinet at 4℃ and stirred for 8 hours. Dialysis and freeze-drying: After stirring, the reaction solution was placed into a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and dialyzed with 3L of deionized water more than 6 times, with an interval of 6-8 hours between each dialysis; after dialysis, the product was freeze-dried to obtain CS-NAC; the freeze-dried product was stored in a sealed container at 4℃ for later use. (2) Preparation of thiolated chitosan CS-NAC-DA coupled with dopa and acetylcysteine: CS-NAC dissolution: Weigh 500mg of CS-NAC into a 200mL Erlenmeyer flask, add 50mL of deionized water solution, and stir for 1h to allow it to swell fully; Preparation of DOPA / DA solution: Weigh 1500 mg of DA solid into a 50 mL Erlenmeyer flask, add 50 mL of deionized water, dissolve at room temperature, and set aside. Preparation of EDC and NHS solution: Weigh 1500 mg of EDC and 750 mg of NHS into a 50 mL Erlenmeyer flask, add 20 mL of deionized water, dissolve at room temperature, and set aside. Carboxyl activation: The EDC and NHS solutions were removed and slowly added dropwise to the DA solution under stirring. After the addition was complete, the mixture was stirred at room temperature for half an hour to activate the carboxyl groups. Coupling reaction and pH adjustment: The CS-NAC solution was slowly added dropwise to the mixed solution of EDC, NHS and DA under stirring. After the addition was complete, the mixture was stirred at room temperature for half an hour. After the addition was complete, the mixture was stirred at room temperature for another half hour. Then, the pH was adjusted by adding 1M sodium hydroxide solution dropwise, and the reaction solution and pH meter count were observed. After the count stabilized at 5.8-6.0, the addition of sodium hydroxide solution was stopped, and the mixed solution was placed in a chromatography cabinet at 4℃ and stirred for 48 hours. Dialysis and freeze-drying: The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyzed with 3L of deionized water more than 6 times, with an interval of 6-8 hours between each dialysis. After dialysis, the product was freeze-dried to obtain CS-NAC-DA. The freeze-dried product was stored in a sealed container at 4℃ for later use. (3) Preparation of the base layer: Step 1: Prepare 20 mL of 2% sodium alginate solution and 12% acrylamide solution using HBSS buffer. Stir on a magnetic stirrer until the solid is completely dissolved, and let stand to remove air bubbles. Step 2: Prepare appropriate amounts of 2% methylenebisacrylamide (MBAA) solution, 0.375M calcium sulfate (CaSO4) solution, and 0.27M ammonium persulfate (APS) solution using HBSS buffer. Step 3: Add 72 μL MBAA solution, 16 μL tetramethylethylenediamine (TEMED) and 400 μL CaSO4 solution dropwise to the sodium alginate and acrylamide mixed solution under high-speed stirring, and stir until the mixture is homogeneous. Step 4: Quickly add 500 μL of APS solution to the mixed solution and stir rapidly for 2 minutes; Step 5: Pour the mixed solution into the mold and let it stand overnight to allow it to crosslink, thus obtaining the base layer; (4) Preparation of novel bioadhesives: Prepare a 4% CS-NAC-DA gel, use a 1mL syringe to slowly drip an appropriate amount onto the surface of the basal layer, and spread it evenly. Cut it into appropriate sizes as needed to obtain a novel bioadhesive.

7. The use of the bioadhesive of claim 1 in the preparation of a medicament for the repair of peripheral nerve injuries, tendon injuries, vascular injuries, and surgical incisions.

8. The use of the bioadhesive of claim 1 in the preparation of a medical material for preventing adhesions after peripheral nerve injury repair surgery.

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