A photoinitiating biological tissue adhesive, gel sheet, preparation method and application

Abstract

The present invention discloses a photoinitiating biological tissue adhesive, a gel sheet, a preparation method and an application thereof. The biological tissue adhesive is cured by a bifunctional polymer and a photoinitiator under photoinitiation; two active units, namely catechol groups and olefin groups, are simultaneously introduced onto a natural polymer through an amidation reaction to prepare the bifunctional polymer. The mixed solution of the bifunctional polymer and the photoinitiator can achieve gelation and appropriate mechanical strength under photo-triggering. By adjusting the grafting ratio of the two active units on the natural polymer, the tissue adhesion strength and the mechanical strength of the adhesive itself are respectively achieved and regulated, so that the adhesive of the present invention can be applied not only to the repair of dura mater (spinal cord), but also to the repair of other tissue traumas.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology, and particularly relates to a photo-initiated biological tissue adhesive, a gel sheet, a preparation method and an application thereof. Background Art

[0002] The dura mater (spinal dura mater) is the outermost layer of the membranes covering the brain and spinal cord, and is a connective tissue fibrous membrane. As a protective structure, when the dura mater (spinal dura mater) is damaged, it is often accompanied by cerebrospinal fluid leakage, that is, the phenomenon that cerebrospinal fluid leaks out from the dura mater (spinal dura mater) gap and the bone defect covered by it under the action of the pressure gradient inside and outside the skull and spinal canal. Continuous cerebrospinal fluid leakage will not only cause disorders in the cerebrospinal fluid circulation dynamics, resulting in orthostatic headache and dizziness, but also further increase the risks of complications such as nervous system infection, poor wound healing, and even intracranial and spinal canal hemorrhage. These complications will consume more medical resources and significantly increase the economic burden on patients; even if patients undergo repeated invasive treatments, not only the effect is poor, but as the condition worsens, they may be severely disabled or even die. According to the etiology, the clinical classification of cerebrospinal fluid leakage can be divided into: traumatic cerebrospinal fluid leakage, postoperative cerebrospinal fluid leakage, and idiopathic cerebrospinal fluid leakage, among which postoperative cerebrospinal fluid leakage is one of the common complications in neurosurgery and spinal surgery. Although with the progress of surgical techniques and the application of new skull base repair materials, the incidence of postoperative cerebrospinal fluid leakage has gradually decreased, its effect is still not ideal. Some studies have shown that the complications related to postoperative cerebrospinal fluid leakage after suboccipital craniotomy are as high as 32%, and the incidence of cerebrospinal fluid leakage after spinal surgery is 5% - 13%. The prevention of cerebrospinal fluid leakage is more important than treatment. In addition to various treatment measures during the operation to prevent cerebrospinal fluid leakage, timely repair when the dura mater ruptures during the operation is the key step to prevent postoperative cerebrospinal fluid leakage. Therefore, it is necessary to solve the problem of cerebrospinal fluid leakage caused by the damage or imperfect suture of the dura mater (spinal dura mater) for various clinical reasons, and this technology can perform controllable and strong adhesion repair of the dura mater (spinal dura mater) in a biological wet environment and with irregular shapes.

[0003] A method of tightly suturing the repaired dura mater (spinal dura mater) in the prior art is to use autologous tissues (fat, muscle, fat, bone chips, etc.) or artificial materials for expanded forming suture. It is mainly a surgical strategy and technique, and there is still a possibility that the suture effect is not good or a leak hole is formed again due to re-rupture. Therefore, it is particularly important to repair and close the first barrier of the dura mater (spinal dura mater).

[0004] Another method is to use fibrin adhesives, namely human-derived medical biological protein adhesives (such as HuGuLaiShi) and porcine-derived medical biological protein adhesives (such as AnKeJing, BeiXiuJiao, etc.), and sealants (such as hydrogels, composite adhesives, etc.). However, this method is only a simple adhesion and filling, with low viscosity; it is easily washed and displaced, resulting in poor sealing effect; especially for high-flow cerebrospinal fluid leakage and cerebrospinal fluid leakage involving the skull base or irregular areas, the sealing effect is not significant; therefore, this method cannot effectively and controllably adhere to the defect boundary, especially in high-flow areas, which is uncontrollable and cannot ensure effective adhesion. Summary of the Invention

[0005] In view of the above problems, the present invention provides a controllable photo-initiated biological tissue adhesive, gel sheet, preparation method and application. By amidation reaction, two active units, catechol group and olefin group, are simultaneously introduced onto the carboxylic acid-containing natural polymer to obtain a bifunctional polymer. The grafting ratio of the two active units on the natural polymer can be adjusted as needed to respectively achieve and control the tissue adhesion strength and the mechanical strength of the adhesive itself. The mixed solution of the bifunctional polymer and the photo-initiator can be gelated and have appropriate mechanical strength under light triggering.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A photo-initiated biological tissue adhesive, wherein the biological tissue adhesive is cured by light irradiation of a bifunctional polymer and a photo-initiator; the bifunctional polymer is obtained by introducing a catechol group and a double bond group into a carboxylic acid-containing natural polymer through an amidation reaction.

[0008] Natural polymers are polymers from nature, such as starch, cellulose, lignin, etc. are all natural polymers. In this application, the natural polymer in the photo-initiated biological tissue adhesive is a carboxylic acid-containing natural polymer with a molecular weight of 1000 - 100000 Da, specifically, it can be natural polymers such as sodium carboxymethyl cellulose, sodium carboxymethyl cellulose, hyaluronic acid, sodium hyaluronate, sodium alginate, carboxymethyl chitin or carboxymethyl chitosan.

[0009] Preferably, the bifunctional polymer is specifically obtained by a condensation reaction of a natural polymer, an amino-containing catechol, an amino-containing olefin and an amidation condensing agent.

[0010] Preferably, the mass ratio of the natural polymer, the amino-containing catechol, the amino-containing olefin and the amidation condensing agent is 1:0.005 - 20:0.005 - 20:0.1 - 1000. Within this range, the biological tissue adhesive can obtain a hydrogel system with good adhesion effect and mechanical strength after curing.

[0011] Preferably, the amino-containing catechol is selected from dopamine, 6-hydroxydopamine, 5-hydroxydopamine, 3,4-dihydroxybenzylamine, norepinephrine, norepinephrine hydrochloride, 3,4-dihydroxynorephedrine or lyciumin B.

[0012] Preferably, the amino-containing olefin is selected from 2-amino methacrylate, 3-buten-1-amine, 2-methylallylamine, methacrylamide, pent-4-ene-1-amine or 4-vinylbenzylamine.

[0013] Preferably, the amidation condensing agent is selected from: 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, active esters (such as N,N'-carbonyldiimidazole, etc.), carbodiimides (such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCl), etc.), onium salts (HATU-PF6, HCTU-PF6, BOP, etc.), organophosphorus compounds (DPPCl, MPTA, BOP-Cl, etc.) and others (such as triphenylphosphine-polyhalomethane, triphenylphosphine-hexachloroacetone, triphenylphosphine-NBS, 3-acyl-2-thiazoline, etc.).

[0014] Preferably, the photoinitiator is selected from any one or several of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, aryl ketone photoinitiators, polycyclic aromatic hydrocarbon photoinitiators, polysilane photoinitiators, acylphosphonate photoinitiators, azo photoinitiators or organometallic complexes.

[0015] Preferably, the mass ratio of the bifunctional polymer to the photoinitiator is 1:0.01 - 10.

[0016] Based on the same inventive concept, the present invention also provides a preparation method of a photoinitiating biological tissue adhesive, comprising the following steps:

[0017] S1: Dissolve the carboxylic acid-containing natural polymer in a solvent to prepare a natural polymer solution;

[0018] S2: Add the amidation reagent into the natural polymer solution prepared in the step S1, then add the amino-containing catechol and the amino-containing olefin in the dark, and adjust the pH of the above solution to 5.5 - 7.5 with NaOH solution to obtain a reaction solution;

[0019] S3: Precipitate the reaction solution obtained in the step S2 in ice ethanol and centrifuge at low temperature, repeat this step 2 - 3 times to obtain a bifunctional polymer;

[0020] S4: After dissolving the bifunctional polymer obtained in the step S3, add a photoinitiator and dissolve it to obtain the biological tissue adhesive.

[0021] When the biological tissue adhesive is irradiated with light, it causes crosslinking and curing of the bifunctional polymer and the photoinitiator.

[0022] Preferably, the solvent in step S1 is one or a mixture of water, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), etc.

[0023] Preferably, the mass ratio of the carboxylic acid-containing natural polymer to the solvent is 1:0.1 - 1000.

[0024] Preferably, the wavelength of the light during the crosslinking reaction of the bifunctional polymer and the photoinitiator is 100 - 1000 nm, and the irradiation time is 1 - 600 s.

[0025] Based on the same inventive concept, the present invention also provides a biological tissue gel sheet. The biological tissue gel sheet is the photoinitiating biological tissue adhesive of the above embodiment or the gel obtained by photoinitiating and curing the photoinitiating biological tissue adhesive obtained by the preparation method, and is trimmed according to the combined requirements to obtain a corresponding sheet product, such as a sheet product with a length, width, and height of 4 cm * 5 cm * 0.5 cm or 8 cm * 10 cm * 0.5 cm.

[0026] Based on the same inventive concept, the present invention also provides an application of the photoinitiating biological tissue adhesive in the preparation of biological adhesion products. The photoinitiating biological tissue adhesive is the photoinitiating biological tissue adhesive described above or obtained by the preparation method.

[0027] Furthermore, the photoinitiating biological tissue adhesive is applied to the repair of dura mater (spinal cord), peritoneum and pleura, the repair of vascular damage, and the adhesion repair of skin and mucosa.

[0028] Furthermore, the form of the biological adhesion product can be an adhesive with a certain fluidity directly packaged in a syringe gun, or a cured gel sheet.

[0029] Due to the adoption of the above technical solutions, the present invention has the following advantages and positive effects compared with the prior art:

[0030] The biological tissue adhesive provided by the present invention is based on a photo-responsive bifunctional natural polymer. The bifunctional natural polymer is prepared by introducing two active units, namely catechol groups and olefin groups, onto a natural polymer containing carboxylic acid through an amidation reaction. Then, under photo-initiation, the bifunctional polymer and the photo-initiator can achieve the curing of the adhesive and its adhesion to biological tissues. Therefore, this tissue adhesive is composed only of a carboxylic acid-modified natural polymer and a photo-initiator, without other small molecule reagents, which can greatly reduce the possibility of tissue inflammation. Moreover, the biological safety and degradability of the natural polymer also provide strong safety guarantees for tissue applications. At the same time, this bifunctional polymer can be easily and efficiently gelated by photo-initiation, with time and space controllability, and is convenient and flexible to use. By regulating the grafting ratio of the two active units, namely catechol groups and olefin groups, on the natural polymer, the required biological tissue adhesion strength and the mechanical strength of the adhesive itself can be achieved and regulated, which is suitable for patching leaks in the dura mater (spinal cord).

[0031] The preparation method system of the biological tissue adhesive of the present invention is simple and highly adjustable. The bifunctional polymer only requires one-step chemical synthesis, the preparation process and components are simple, and the specific structures and grafting ratios of the two active components are highly adjustable. Description of the Drawings

[0032] Figure 1 It is a test chart of the toxicity levels of biological tissue adhesives with different volume concentrations in microglia (BV2) in Example 1 of the present invention;

[0033] Figure 2 It is the rheological behavior of the biological tissue adhesive in Example 1 of the present invention before photo-crosslinking;

[0034] Figure 3 It is the rheological behavior of the biological tissue adhesive in Example 1 of the present invention after photo-crosslinking;

[0035] Figure 4 It is a closed cerebrospinal fluid biological tissue model constructed with the swim bladder of the present invention. Using the biological tissue adhesive in Example 1 to block the leak (a cut of about 1.5 cm was made with a blade), after injecting physiological saline, the liquid oozed and leaked significantly from the leak. Then, after photo-triggering the cross-linking and adhesion of the adhesive for 15 seconds, the gap was repaired. By injecting water into the biological tissue model to simulate the cerebrospinal fluid filling effect, it was found that there was no liquid oozing or leaking at the leak after complete water injection, and the repair effect was good;

[0036] Figure 5For the closed cerebrospinal fluid biological tissue model constructed with the swim bladder of the present invention, 15 seconds after using the biological tissue adhesive in Example 1 to seal the leak, it was observed that there was no liquid exudation or leakage at the leak after complete water injection. Then, a limit bursting pressure of 25 - 35 cmH2O was applied by hand to simulate the transient increase in intracranial pressure during postoperative body position changes, sneezing, coughing, and vomiting. It was seen that the adhesive swelled as the break opened, and no normal saline leaked out, demonstrating good adhesion and mechanical elasticity;

[0037] Figure 6 For the closed cerebrospinal fluid biological tissue model constructed with the porcine intestinal mucosa of the present invention, the biological tissue adhesive in Example 1 was used to seal the leak (a cut of about 1.5 cm was made with a blade). After injecting normal saline, obvious liquid exudation and leakage occurred from the leak. Then, the adhesive was crosslinked and bonded by light triggering for 15 seconds to repair the gap. Water was injected into the biological tissue model to simulate the cerebrospinal fluid filling effect. It was observed that there was no liquid exudation or leakage at the leak after complete water injection, and the repair effect was good, verifying the biological effectiveness of the adhesive from various biological tissues;

[0038] Figure 7 For the cerebrospinal fluid leakage rat model constructed by the present invention, the biological tissue adhesive and normal saline in Example 1 were used to repair the cerebrospinal fluid leakage animal model of rats, Figure 7 where A is to observe the wound healing and cerebrospinal fluid leakage conditions; Figure 7 where C is the change in body weight of each group after feeding; Figure 7 where B is the overall HE - stained tissue section of the rat scalp, galea aponeurotica, dura mater, arachnoid mater, and pia mater after sudden death and tissue sampling;

[0039] Figure 8 The pictures show the cell growth morphology under a light microscope in the BV2 microglial cell line culture system with the addition of the biological tissue adhesive diluted to a volume concentration of 6.3% and the normal saline blank control group respectively. Detailed implementation manners

[0040] Current tissue adhesives provide a variety of materials for wound management and are widely used in various medical settings, from minor to life - threatening tissue injuries. Compared with traditional wound - closing methods (i.e., suturing and stapling), tissue adhesives are relatively easy to use, can be applied quickly, and minimize tissue damage. In addition, tissue adhesives can be used as hemostatic agents to control bleeding and provide a tissue - healing environment at the wound site. An ideal tissue adhesive should have many properties, including: (1) biocompatibility and non - toxicity; (2) the ability to form strong interactions with tissues; (3) mechanical similarity to the adhered tissues; (4) mechanical ability to withstand repeated tissue stress; (5) acceptable swelling properties to minimize tissue compression; and (6) biodegradability compatible with the tissue - healing rate.

[0041] However, existing adhesives only meet some of these requirements and still face some limitations and unresolved challenges (e.g., weak adhesion strength, poor mechanical properties, complex systems, low biosecurity, inflammation, etc.), which limit their use and leave room for further improvement.

[0042] Since there is cerebrospinal fluid in the dura mater (spinal cord), the adhesive has a large adhesive force in a wet or liquid environment and needs to maintain a good sealing effect under the internal and external pressures of the skull and spinal canal. In view of the problems existing in the current dura mater (spinal cord) repair, the present invention designs and prepares a photoinitiating biological tissue adhesive, which includes a bifunctional polymer. The mixed solution of the bifunctional polymer and the photoinitiator can be gelated under photoinitiation, and the gel after gelation has excellent mechanical strength. The bifunctional polymer of the present invention simultaneously introduces catechol units and olefin units onto a natural polymer containing carboxylic acid through an amidation reaction. The natural polymer has excellent biocompatibility and degradability, providing a strong safety guarantee for use as a cerebrospinal membrane repair. After the adhesive is applied for tissue adhesion, it can fuse with the tissue and degrade; the catechol units and olefin units provide binding biological sites for the adhesive and the tissue to be repaired, enabling the adhesive to have excellent mechanical properties. In particular, the catechol units have an efficient chemical interaction with the amino groups or sulfhydryl groups abundantly present in biological tissues to obtain stable chemical bonds, so that it also has a high bonding strength in a wet environment, and the repaired break will not be damaged under greater pressure; the bifunctional polymer can be conveniently and efficiently gelated by photoinitiation, with time and space controllability and convenient and flexible use. The present invention can also adjust the grafting ratio of the two active units on the natural polymer as needed to achieve and control the subsequent tissue adhesion strength and the mechanical strength of the adhesive, so that the adhesive provided by the present invention is not only suitable for dura mater (spinal cord) repair, but also applicable to other tissue traumas, such as the repair of peritoneum and pleura, the repair of blood vessel damage, the adhesion repair of skin and mucosa, etc.

[0043] The following further elaborates in detail on a photoinitiating biological tissue adhesive, a gel sheet, a preparation method and an application thereof proposed by the present invention with reference to the accompanying drawings and specific embodiments. According to the following description, the advantages and features of the present invention will be clearer.

[0044] Example 1

[0045] S1: Add 2.0 g of sodium carboxymethylcellulose to 200 mL of deionized water and stir at room temperature to obtain a sodium carboxymethylcellulose solution;

[0046] S2: Add 1.38 g of amidation reagent 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride to the sodium carboxymethylcellulose solution in step S1 and dissolve to obtain a homogeneous solution; add 114 mg of dopamine hydrochloride and 100 mg of 2-aminoethyl methacrylate hydrochloride to the above mixed solution and stir in the dark until completely dissolved; adjust the pH of the above solution to 6.5 with 0.5 M NaOH solution and stir at room temperature for 24 h to obtain a reaction solution;

[0047] S3: Precipitate the reaction solution obtained in step S2 in 800 mL of ice ethanol and centrifuge at low temperature to obtain a polymer; dissolve the polymer again in 200 mL of deionized water and precipitate again in ice ethanol, and obtain a bifunctional polymer by low-temperature centrifugation and freeze-drying;

[0048] S4: Take 45 mg of the bifunctional polymer and dissolve it in 3 mL of deionized water to obtain a bifunctional polymer solution, add 6 mg of photoinitiator lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, and shake or stir to completely dissolve it to obtain a biological tissue adhesive; irradiate the obtained biological tissue adhesive with a 365 nm ultraviolet lamp for 2 s to complete the photocrosslinking process and obtain a cured biological tissue adhesive.

[0049] 1. Cytotoxicity test

[0050] The CCK-8 kit was selected to test the cytotoxicity of the obtained tissue adhesive. The CCK-8 kit is a rapid, highly sensitive, and non-radioactive colorimetric detection kit based on WST-8 and widely used in cell proliferation and cytotoxicity tests. The CCK-8 solution can be directly added to cell samples without pre-preparing various components. In the presence of an electron-coupling reagent, WST-8 can be reduced by some dehydrogenases in mitochondria to generate orange-yellow formazan. The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of color (the amount of formazan generated) is linearly related to the number of cells. WST-8 is an upgraded alternative to MTT and has obvious advantages compared with MTT or other MTT-like products such as XTT and MTS. First, the formazan generated by the reduction of MTT by some dehydrogenases in mitochondria is not water-soluble and requires a specific solvent to dissolve; while the formazan generated by WST-8, XTT, and MTS is water-soluble, eliminating the subsequent dissolution step. Second, the formazan generated by WST-8 is more soluble than that generated by XTT and MTS. Third, WST-8 is more stable than XTT and MTS, making the experimental results more reliable. Fourth, WST-8 has a wider linear range, higher sensitivity, and greater stability compared with MTT, XTT, etc. WST-8 has no obvious toxicity to cells. After adding the CCK-8 solution for color development, the microplate reader can be used to read the plate repeatedly at different times, and the detection time is more flexible, facilitating the determination of the optimal measurement time.

[0051] (1) Preparation of the standard curve

[0052] 1. Inoculate 100 μL of BV2 (microglial) cell suspension (5000 cells / well) into a 96-well plate;

[0053] 2. Dilute it proportionally with the culture medium in an equal ratio to form a cell concentration gradient. Generally, 5-7 cell concentration gradients are required, with 4-6 replicates in each group;

[0054] 3. After inoculation, culture for 2-4 hours to allow the cells to adhere, and then add 10 μL of CCK-8 reagent to every 100 μL of culture medium. After culturing for a certain time, measure the OD value to prepare a standard curve with the cell number as the abscissa and the OD value as the ordinate. The cell number of the unknown sample can be determined according to this standard curve. The prerequisite for using this standard curve is that the experimental conditions are completely consistent.

[0055] (2) Cell proliferation-cytotoxicity experiment

[0056] 1. Inoculate 100 μL of cell suspension (5000 cells / well) into a 96-well plate;

[0057] 2. Incubate in a pre-placed 37°C incubator with 5% CO2 and saturated humidity for 24 hours;

[0058] 3. Dilute the tissue adhesive with 100 μL of fresh medium to different volume concentrations (50%, 25%, 12.5%, 6.3%, 3.1%, 1.6%, 0.8%, 0.4%, 0.2%), and add it to the culture plate containing cells. The control group only adds microglia medium;

[0059] 4. Incubate in the incubator for 24 hours;

[0060] 5. Dilute the CCK-8 reagent with medium to a 10% concentration and add it to the well plate for 30 min of incubation;

[0061] 6. Measure the absorbance at a wavelength of 450 nm.

[0062] As Figure 1 are the absorbance values of the tissue adhesive at different volume concentrations and the blank control group. It can be seen from the figure that the absorbance values of the tissue adhesive at different volume concentrations are basically greater than those of the control group, except that the absorbance value of 50% is slightly less than that of the control group, indicating that the synthesized biological tissue adhesive has no cytotoxicity; especially the absorbance values of the volume concentrations of 25%, 12.5%, and 6.3% are much greater than those of the blank control group, indicating that the biological tissue adhesive also has a trophic and proliferative promoting effect on glial cells.

[0063] In addition, for the BV2 cell line culture under a light microscope, the experimental group added the biological tissue adhesive diluted to different volume concentrations, and the control group was not added. After culturing in the incubator for 24 hours, observe under the light microscope. As Figure 8 shows the electron micrographs of the experimental group adding the biological tissue adhesive diluted to a 6.3% volume concentration and the control group. The cell morphology of the experimental group and the control group grew well, and the morphology of the experimental group was more plump. Combining with the CCK8 experimental data, it is proved that the hydrogel designed and synthesized by us not only has no cytotoxicity, but also has a certain trophic and proliferative promoting effect on glial cells.

[0064] 2. Whether the cross-linking performance after photoinitiation

[0065] The rheological test of the biological tissue adhesive in Example 1 before and after photo-crosslinking was carried out on a rotational rheometer (Thermo Scientific, Mars 60). The rotational time sweep mode was selected, the temperature was 37°C, the rotational rates were set to 0.01 / s, 0.1 / s, 0.5 / s, 1.0 / s, 5.0 / s, and 10 / s respectively. Each rotational rate was scanned for 2 min, and 100 data points were collected at each rotational rate. Apply the aqueous solution of the bifunctional polymer containing the photo-crosslinking agent (1.5 wt%) on the turntable of the rheometer with a thickness greater than 1 mm, and then the rheological behavior of the adhesive before photo-crosslinking can be tested. AsFigure 2 As shown, the polymer solution applied on the frustum of a cone is subjected to photo-crosslinking for about 30 s to achieve in-situ crosslinking, and then the rheological behavior of the crosslinked adhesive can be tested. For example, Figure 3 as shown.

[0066] For example Figure 2 and Figure 3 As can be seen, the loss modulus G" of the sample before crosslinking is greater than its storage modulus G', indicating that the system is in a liquid flow state. After crosslinking, its storage modulus G' is greater than its loss modulus G", and it can remain at a stable value, indicating the formation of an elastic three-dimensional network, which shows that the bifunctional polymer and the photoinitiator undergo a crosslinking reaction and curing after photoinitiation. Indirectly, it also shows that the cured product has bonding strength.

[0067] 3. A simple biological model of cerebrospinal fluid leakage is prepared using fish bladders and intestinal mucosa. As Figure 4 shown, the leaky orifice (a cut about 1.5 cm long made by a blade) is blocked with the biological tissue adhesive of the example. After injecting normal saline, liquid oozes and leaks out significantly from the leaky orifice. Then, after the photo-triggered adhesive crosslinks and bonds for 15 s by the example 1 to repair the gap, water is injected into the biological tissue model to simulate the cerebrospinal fluid filling effect. As Figure 4 shown, it can be seen that there is no liquid oozing or leaking from the leaky orifice after complete water injection, and the repair effect is good. Then, 15 s after the biological tissue adhesive blocks the break or leaky orifice, it can be seen that there is no liquid oozing or leaking from the leaky orifice after complete water injection. Then, a limit bursting pressure of 25 - 35 cmH2O is applied by hand to simulate the transient increase in intracranial pressure during postoperative body position change, sneezing, coughing, and vomiting. It can be seen that the adhesive swells as the break opens, and no normal saline leaks out, demonstrating good adhesion and mechanical elasticity. As Figure 5 shown. Similarly, we select a closed cerebrospinal fluid biological tissue model constructed with porcine intestinal mucosa and conduct the same experiment. After the photo-triggered adhesive crosslinks and bonds for 15 s to repair the gap, water is injected into the biological tissue model to simulate the cerebrospinal fluid filling effect. It can be seen that there is no liquid oozing or leaking from the leaky orifice after complete water injection, and the repair effect is good, verifying the biological effectiveness of the adhesive from multiple biological tissues. As Figure 6 shown. The above further shows that the bonding strength and mechanical strength of the adhesive after photocuring in this example enable it to be applied to the repair of the dura mater (spinal cord).

[0068] 4. Animal experiments to evaluate the effectiveness and biocompatibility of the biological tissue adhesive

[0069] A: Male SD rats weighing about 160 g were selected. The skull was exposed at the right frontal part (5 mm to the right of the sagittal suture and 5 mm in front of the coronal suture), and the dura mater and subarachnoid space were incised to simulate a craniotomy cerebrospinal fluid leakage model. An experimental group and a control group were set up, with 3 rats in each group. After the cerebrospinal fluid leakage rat model was successfully constructed in the experimental group, 1 ml of the biological tissue adhesive of Example 1 was used to cover the wound. After confirming satisfactory closure, light source excitation adhesion fixation was performed, and the wound was sutured; similarly, in the control group, after the cerebrospinal fluid leakage rat model was successfully constructed, 1 ml of normal saline was used to cover the wound. After confirming satisfactory closure, light source excitation adhesion fixation was performed, and the wound was sutured.

[0070] B: After the experiments of the rats in the experimental group and the control group were successful, they were raised for 15 days, and then

[0071] (1) Specimen collection: The heads of the rats in the experimental group and the control group were respectively placed in embedding frames.

[0072] (2) Dehydration and wax infiltration: The collected tissues together with the embedding frames were taken out and placed in a dehydration basket. They were dehydrated in a dehydrator with gradient ethanol in turn: 75% ethanol for 2 h - 85% ethanol for 2 h - 90% ethanol for 1.5 h - 95% ethanol for 2 h - absolute ethanol I for 2 h - absolute ethanol II for 2 h - ethanol-benzene for 40 min - xylene I for 40 min - xylene II for 40 min - 65°C; then wax infiltration: molten paraffin I for 0.5 h - 65°C, molten paraffin II for 1 h - 65°C, molten paraffin III for 2 h 45 min.

[0073] (3) Embedding: The tissues infiltrated with wax were embedded in an embedding machine. First, the melted wax was put into the embedding frame. Before the wax solidified, the tissues were taken out of the dehydration box and placed in the embedding frame according to the requirements of the embedding surface and the corresponding labels were pasted. It was cooled on a -20°C freezing table. After the wax solidified, the wax block was taken out of the embedding frame and the wax block was trimmed.

[0074] (4) Sectioning: Along the exposed part of the experimental skull, biological sections such as skin, muscle, dura mater, arachnoid and cortical brain tissue were vertically cut in full layer. The trimmed wax block was placed on a paraffin slicer for sectioning, with a thickness of 4 μm. The sections were floated on the warm water of a spreading machine at 40°C to flatten the tissues, and the tissues were picked up with glass slides and baked in an oven at 60°C. After the water was dried and the wax was melted, it was taken out and stored at room temperature for standby.

[0075] (5) Staining: Staining was performed using the classical hematoxylin-eosin staining method (HE), as Figure 7In B, the control group and the experimental group respectively showed the complete structures of the skin, muscle, dura mater, arachnoid mater, and cortical brain tissue. The focus was on observing the cortical brain tissue and the skin and muscle tissues under 10x and 20x microscopes. The results showed that: there were no inflammatory, necrotic, hemorrhagic, or abnormal hyperplastic manifestations in the skin, muscle, dura mater, arachnoid mater, and cortical brain tissue of the experimental group and the control group. Moreover, the bioadhesive was basically absorbed, and there was no residue or foreign body encapsulation, indicating that the bioadhesive had good biocompatibility and degradability.

[0076] C: After the experiments on the rats in the experimental group and the control group were successful, they were raised for 15 days. The wounds of the two groups of rats were observed at time points of 1, 3, 5, 7, 9, 11, 13, and 15 days. There were no abnormal conditions such as wound swelling and infection in the experimental group and the control group. A small amount of cerebrospinal fluid leakage occurred in the control group, while no cerebrospinal fluid leakage was observed in the experimental group. The weight changes of the two groups of rats were detected. As Figure 7 In C, there was no statistical difference in the weight curves between the experimental group and the control group, proving that the weight development of the rats in the experimental group was good and further proving good biocompatibility.

[0077] Example 2

[0078] S1: Add 3.0 g of sodium carboxymethylcellulose to 200 mL of deionized water and stir at room temperature to obtain a sodium carboxymethylcellulose solution;

[0079] S2: Add 2.76 g of amidation reagent bis(2-oxo-3-oxazolidinyl)phosphoryl chloride to the sodium carboxymethylcellulose solution in step S1 and dissolve to obtain a homogeneous mixed solution; add 200 mg of norepinephrine and 150 mg of 3-buten-1-amine to the above mixed solution, and stir in the dark until completely dissolved; adjust the pH of the above solution to 7.0 with 0.5 M NaOH solution and stir at room temperature for 24 h to obtain a reaction solution;

[0080] S3: Precipitate the reaction solution obtained in step S2 in 800 mL of ice ethanol and centrifuge at low temperature to obtain a polymer; dissolve the polymer again in 200 mL of deionized water, precipitate again in ice ethanol, and obtain a bifunctional polymer through low-temperature centrifugation and freeze-drying;

[0081] S4: Take 45 mg of the bifunctional polymer and dissolve it in 3 mL of deionized water to obtain a bifunctional polymer solution. Add 6 mg of photoinitiator 2,5-bis-[4-(diethylamino)-benzylidene]-cyclopentanone, and shake or stir it to completely dissolve to obtain a bio-tissue adhesive; irradiate the obtained bio-tissue adhesive with a laser of wavelength 800 nm for 10 s to complete the photo-crosslinking process and obtain a cured bio-tissue adhesive.

[0082] Example 3

[0083] S1: Add 2.0 g of sodium hyaluronate into a mixed solvent of 120 mL of deionized water and 80 mL of N,N-dimethylformamide, and stir at room temperature to obtain a sodium hyaluronate solution;

[0084] S2: Add 0.69 g of amidation reagent N,N'-carbonyldiimidazole into the sodium hyaluronate solution in step S1 and dissolve to obtain a homogeneous mixed solution; add 57 mg of 5-hydroxydopamine hydrochloride and 50 mg of 4-vinylbenzylamine into the above mixed solution, and stir in the dark until completely dissolved; adjust the pH of the above solution to 6.0 with 0.5 M NaOH solution, and stir at room temperature for 24 h to obtain a reaction solution;

[0085] S3: Precipitate the reaction solution obtained in step S2 in 800 mL of ice ethanol, and centrifuge at low temperature to obtain a polymer; dissolve the polymer in 200 mL of deionized water again, precipitate in ice ethanol again, and obtain a bifunctional polymer through low-temperature centrifugation and freeze-drying;

[0086] S4: Take 30 mg of the bifunctional polymer and dissolve it in 3 mL of deionized water to obtain a bifunctional polymer solution, add 3 mg of photoinitiator phenyl eosin Y, and shake or stir to completely dissolve it to obtain a biological tissue adhesive; irradiate the obtained biological tissue adhesive with 530 nm LED for 20 s to complete the photo-crosslinking process and obtain a cured biological tissue adhesive.

[0087] The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Even if various changes are made to the present invention, provided that these changes fall within the scope of the claims of the present invention and its equivalent technologies, they still fall within the protection scope of the present invention.

Claims

1. Use of a photoinitiating biological tissue adhesive in the preparation of dura mater and spinal dura mater repair products, wherein the biological tissue adhesive is cured by a bifunctional polymer and a photoinitiator under light irradiation; The bifunctional polymer is obtained by simultaneously introducing catechol groups and double bond groups into a carboxylic acid-containing natural polymer through an amidation reaction; The specific preparation method of the bifunctional polymer includes the following steps: S1: Dissolve the carboxylic acid-containing natural polymer in a solvent to prepare a natural polymer solution; S2: Add an amidation reagent to the natural polymer solution prepared in step S1, then add catechol-containing amino groups and olefin-containing amino groups in the dark, and adjust the pH of the above solution to 5.5 - 7.5 with a NaOH solution to obtain a reaction solution; S3: Precipitate the reaction solution obtained in step S2 in ice ethanol and centrifuge at low temperature, repeat this step 2 - 3 times to obtain the bifunctional polymer; The mass ratio of the carboxylic acid-containing natural polymer, catechol-containing amino groups, olefin-containing amino groups, and amidation condensing agent is 1:0.057:0.05:0.69 or 3:0.2:0.15:2.76 or 1:0.0285:0.025:0.345; The olefin-containing amino group is selected from 2-amino methacrylate, 3-butene-1-amine, 2-methylallylamine, pent-4-ene-1-amine, or 4-vinylbenzylamine; The catechol-containing amino group is selected from dopamine, 6-hydroxydopamine, 5-hydroxydopamine, 3,4-dihydroxybenzylamine, norepinephrine, norepinephrine hydrochloride, 3,4-dihydroxy-norephedrine, or lyciumin; The carboxylic acid-containing natural polymer is carboxymethyl cellulose, sodium carboxymethyl cellulose, hyaluronic acid, sodium hyaluronate, sodium alginate, carboxymethyl chitin, or carboxymethyl chitosan; By regulating the grafting ratio of two active units, namely catechol groups and olefin groups, on the natural polymer, the required biological tissue adhesion strength and the mechanical strength of the adhesive itself are realized and regulated, which is suitable for mending leaks in the dura mater and spinal dura mater.

2. Use of the photoinitiating biological tissue adhesive according to claim 1 in the preparation of dura mater and spinal dura mater repair products, characterized in that, The amidation condensing agent is selected from any one of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, active esters, carbodiimides, diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, onium salts, organophosphorus compounds, triphenylphosphine - polyhalomethane, triphenylphosphine - hexachloroacetone, triphenylphosphine - NBS, 3-acyl-2-thiazoline, or nitrosonium tetrafluoroborate; 3. Use of the photoinitiating biological tissue adhesive according to claim 1 in the preparation of dura mater and spinal dura mater repair products, characterized in that, The photoinitiator is selected from any one or several of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, aryl ketone photoinitiators, polycyclic aromatic hydrocarbon photoinitiators, polysilane photoinitiators, acylphosphonate photoinitiators, azo photoinitiators, or metal organic complexes; 4. Use of the photoinitiating biological tissue adhesive according to claim 1 or 3 in the preparation of dura mater and spinal dura mater repair products, characterized in that, The mass ratio of the bifunctional polymer to the photoinitiator is 1:0.01 - 10.

5. Use of the photoinitiating biological tissue adhesive according to claim 1 in the preparation of dura mater and spinal dura mater repair products, characterized in that, The repair product is a sheet product.

6. Use of the photoinitiating biological tissue adhesive according to claim 1 in the preparation of dura mater and spinal dura mater repair products, characterized in that, The preparation of the biological tissue adhesive includes dissolving the bifunctional polymer and then adding a photoinitiator to dissolve, to obtain the biological tissue adhesive; under light irradiation, the bifunctional polymer and the photoinitiator in the biological tissue adhesive undergo a crosslinking reaction and cure.

7. Use of the photoinitiating tissue adhesive according to claim 1 in the preparation of dura mater and spinal dura mater repair products, characterized in that, The solvent in the step S1 is one or a mixture of water, N,N-dimethylformamide or dimethyl sulfoxide.

8. Use of the photoinitiating biological tissue adhesive according to claim 1 or 7 in the preparation of dura mater and spinal dura mater repair products, characterized in that, The mass ratio of the carboxylic acid-containing natural polymer to the solvent is 3:200 - 3000.

9. Use of the photoinitiating biological tissue adhesive according to claim 6 in the preparation of dura mater and spinal dura mater repair products, characterized in that, When the bifunctional polymer and the photoinitiator undergo a crosslinking reaction under light irradiation, the wavelength of the light is 365 - 1000 nm, and the irradiation time is 1 - 600 s.

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