Oral repair membrane material and method of making same

Abstract

The application relates to the technical field of oral biological medical materials, in particular to an oral repair membrane material and a preparation method thereof. In order to improve the antibacterial performance of the oral repair membrane, the prepared oral repair membrane material adopts a multilayer composite membrane structure, the material of a back membrane in contact with the external oral environment is a membrane prepared from PLGA, and the inside uses a gelatin-based hydrogel with an antibacterial function as the inner layer material of the oral repair membrane, wherein thiofene and imidazole groups are contained, bacteria of common periodontal diseases can be inhibited, and therefore the alveolar bone can be prevented from being invaded by gingival porphyromonas in the repair process, and the repair process is accelerated.

Description

Technical Field

[0001] This invention relates to the field of oral biomedical materials technology, specifically to an oral repair membrane material and its preparation method. Background Technology

[0002] As the beginning of the human digestive tract, the oral cavity is responsible for chewing and eating, with teeth playing a major role in the chewing process. However, due to periodontitis, external trauma, and poor oral hygiene habits, teeth are often severely damaged, making tooth loss a common clinical disease. Although dental implants are a solution, the alveolar bone often atrophies due to the lack of external functional stimulation before implantation, resulting in insufficient bone volume for implantation. Therefore, in clinical practice, oral repair membranes are often used to create a biological barrier in the implantation area, combined with bone powder to promote bone growth. This selectively prevents faster-growing gingival soft tissue cells from prematurely invading the bone defect area, creating a protected regeneration space for slower-growing bone cells, guiding bone tissue repair, and ensuring stable implantation.

[0003] However, during the dental implantation process, the presence of a large number of microorganisms in the oral cavity can easily lead to infection of the implantation wound, which in turn affects the implantation site, hindering the osteogenic function of osteoblasts and preventing bone growth, thus resulting in implantation failure. Therefore, it is necessary to address this deficiency by giving the oral repair membrane certain antibacterial and bacteriostatic functions, thereby improving the implantation success rate. Summary of the Invention

[0004] The purpose of this invention is to provide an oral repair membrane material and its preparation method, which endows the oral repair membrane material with certain antibacterial properties to solve the bone repair function of the oral repair membrane and improve the implantation success rate.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing an oral repair membrane material, comprising the following steps:

[0007] S1. Mix antibacterial crosslinkable modified gelatin with methacrylamide gelatin, add the mixture to PBS buffer, heat and stir until completely dissolved, add hydroxyapatite and photoinitiator, sonicate and mix evenly, add to molding container, use ultraviolet light to mold, and obtain antibacterial hydrogel.

[0008] The preparation method of the antibacterial crosslinkable modified gelatin includes the following steps:

[0009] a. Methacrylic acid is dissolved and then reacted with thionyl chloride to prepare a methacryloyl chloride intermediate;

[0010] b. Dissolve 1H-imidazol-4,5-diethanol in triethylamine and then react it with methacryloyl chloride intermediate to obtain the imidazolium intermediate;

[0011] c. Mix 3-thiophene malonic acid with deionized water, add NHS and EDC·HCl, stir to activate, then add dropwise anhydrous ethanol solution containing imidazole intermediate, stir to react, and obtain imidazole-thiophene intermediate;

[0012] d. Mix gelatin with PBS buffer, add imidazole-thiophene intermediate, hydroquinone, EDC·HCl and NHS, stir and react to obtain antibacterial crosslinkable modified gelatin;

[0013] S2. After attaching the antibacterial hydrogel to the inner side of the back membrane, cut it into shape, and dry it to obtain the oral repair membrane material.

[0014] Furthermore, in step S1, the preparation method of the antibacterial crosslinkable modified gelatin specifically includes the following steps:

[0015] a. Dissolve methacrylic acid in anhydrous dichloromethane, disperse and mix evenly, add thionyl chloride and mix evenly, add DMF, under nitrogen atmosphere protection, heat and reflux and stir for 4-6 hours, remove excess solvent by rotary evaporation to obtain methacryloyl chloride intermediate;

[0016] b. Dissolve 1H-imidazol-4,5-diethanol and triethylamine in anhydrous dichloromethane, mix thoroughly, cool to a constant temperature in an ice-water bath, and add dropwise an anhydrous dichloromethane solution containing the methacryloyl chloride intermediate prepared in step a), stirring continuously during the dropwise addition. After the dropwise addition is completed, heat the reaction system and stir to react. Wash the resulting reaction solution 2-3 times with saturated sodium chloride solution and deionized water, collect the organic phase, dry it overnight with anhydrous sodium sulfate, filter, and vacuum dry the filtrate to obtain the imidazolium intermediate.

[0017] c. Disperse the imidazole intermediate in anhydrous ethanol, and stir until the dispersion is uniform to obtain an anhydrous ethanol solution containing the imidazole intermediate;

[0018] 3-Thiophene malonic acid was added to deionized water, and NHS and EDC·HCl were added to it. After activation by stirring in the dark, an anhydrous ethanol solution containing the imidazole intermediate was added dropwise. After the addition was completed, the reaction was stirred at room temperature for 12-18 hours, and the pH of the reaction system was controlled at 6-7 during the stirring process. After the reaction was completed, hydrochloric acid was added dropwise to the reaction solution to adjust the pH of the solution to 1.5-2. After stirring for 0.5-1 hour, the precipitate was separated by filtration, washed with deionized water 3-5 times, and dried to obtain the imidazole-thiophene intermediate.

[0019] d. Mix gelatin with PBS buffer and stir until the gelatin is completely dissolved. Cool to room temperature, add imidazole-thiophene intermediate, hydroquinone, EDC·HCl and NHS. Heat and stir to react. Dilute with 3-4 times the volume of PBS buffer and deionized water to stop the reaction. Dialyze the mixture for 3-5 days, and freeze-dry the product in the dialysis bag to obtain antibacterial crosslinkable modified gelatin.

[0020] Furthermore, in step a, the mass ratio of methacrylic acid, sulfoxide, and DMF is 1:(1.3~1.6):(0.05~0.1).

[0021] When heating, the heating temperature is 38~40℃.

[0022] Furthermore, in step b, the mass ratio of 1H-imidazol-4,5-diethanol, triethylamine, and methacryloyl chloride intermediate is 1:(0.8~1.3):(0.7~0.9).

[0023] When the reaction system is heated and stirred, the heating temperature is 24~26℃ and the reaction time is 6~8h.

[0024] Furthermore, in step c, the mass ratio of 3-thiophene malonic acid, NHS, EDC·HCl, and imidazole intermediate is 1:(0.6~0.65):(1~1.1):(1~1.2).

[0025] Furthermore, in step d, the mass ratio of gelatin, imidazole-thiophene intermediate, hydroquinone, EDC·HCl and NHS is 1:(0.5~0.9):(0.01~0.04):(2.5~4.5):(1.7~3.5).

[0026] Heat to 38~45℃, and stir the reaction. The heating temperature is 38~45℃, and the reaction time is 3~4h.

[0027] Furthermore, in step d, the dialysis bag used during dialysis is 3~5kDa; the dialysis temperature is 2~8℃; and the dialysis fluid is changed every 6~8 hours.

[0028] Furthermore, in step S1, the mass ratio of antibacterial crosslinkable modified gelatin, methacrylamide gelatin, hydroxyapatite, and photoinitiator is (1~2):(8~9):(3~5):(0.15~0.35).

[0029] Furthermore, in step S1, the temperature is raised to 48~52℃ and irradiated with ultraviolet light for 65±5s; in step S2, the back film is a PLGA film.

[0030] Secondly, the present invention provides an oral repair membrane material, which is prepared by the above method.

[0031] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0032] To enhance the antibacterial properties of oral repair membranes, this invention improves the materials used in the oral repair membrane. The oral repair membrane material prepared in this invention adopts a multi-layer composite membrane structure. The back membrane, which comes into contact with the external oral environment, is made of PLGA. Compared with conventional materials such as gelatin and hydrogel, PLGA has a longer degradation cycle, which can reach 2-3 months. This can better match the osteoblast proliferation cycle and avoid the defects of failure to degrade or excessively long degradation time, which may affect implantation.

[0033] Furthermore, this invention further prepares a gelatin-based hydrogel with antibacterial function as the inner layer material of an oral repair membrane. Because conventional gelatin materials have insufficient cross-linking, this invention performs cross-linking treatment on the gelatin material, and in this process, it also performs antibacterial treatment. This invention first uses methacrylic acid as a raw material, treating it with sulfoxide to achieve acyl chloride treatment of the carboxyl groups, thereby reducing the activation energy of the reaction with 1H-imidazol-4,5-diethanol in step b, allowing it to proceed at a lower temperature and avoiding self-crosslinking of double bonds caused by high temperatures; subsequently, this invention further prepares the acyl-chlorinated methacryloyl chloride... The intermediate was reacted with 1H-imidazol-4,5-diethanol and mixed, and by controlling the reaction conditions, methacryloyl chloride was allowed to react with one of the alcohol hydroxyl groups in 1H-imidazol-4,5-diethanol, thereby preparing an imidazolium intermediate with an imidazolium ring, a double bond, and a free alcohol hydroxyl group. The imidazolium ring is a five-membered heterocycle containing two nitrogen atoms, in which the nitrogen atoms have lone pairs of electrons. Under physiological conditions, these nitrogen atoms can be partially protonated to generate cations. Since bacterial cell membranes usually contain anionic groups such as lipopolysaccharides, they can combine with the imidazolium ring, thereby disrupting the integrity of the cell membrane and thus achieving antibacterial properties against pathogens.

[0034] During the alveolar bone repair process, the main pathogen causing periodontitis and other related conditions is Porphyromonas gingivalis. As a typical Gram-negative anaerobic bacterium, its surface contains a large amount of lipopolysaccharide, with a large number of phosphate and carboxylic acid groups. Its anion density is much higher than that of Gram-positive bacteria. Therefore, when faced with imidazole rings, the bacterial activity of Porphyromonas gingivalis is severely inhibited, thus protecting the alveolar bone from invasion by Porphyromonas gingivalis during the repair process and accelerating the repair process.

[0035] Subsequently, this invention further utilizes 3-thiophene malonic acid as a raw material. Under the catalytic activation of NHS and EDC·HCl catalysts, the carboxyl group in 3-thiophene malonic acid is activated, thereby reducing the activation energy of the reaction with the hydroxyl group in the imidazole intermediate, lowering the reaction temperature, and further avoiding premature cross-linking of the double bond. Furthermore, after the reaction of 3-thiophene malonic acid with the imidazole intermediate is completed, the resulting imidazole-thiophene intermediate is an organic compound with a carboxyl group end cap, exhibiting weak acidity. When the pH is adjusted to 2.0 using dilute hydrochloric acid, the carboxyl group is protonated, resulting in a sharp decrease in the water solubility of the product, allowing it to precipitate. This process can also effectively remove the water-soluble unreacted raw materials from the aforementioned reaction, thereby reducing the problem of excessive biotoxicity of the subsequently prepared hydrogel.

[0036] Furthermore, the thiophene ring introduced in this step is a five-membered heterocycle containing sulfur atoms. The sulfur atoms in it have low electronegativity and relatively high hydrophobicity, which allows it to insert into the phospholipid bilayer of the bacterial cell membrane, thereby interfering with cell membrane activity. Therefore, unlike the imidazole ring, the antibacterial properties of the thiophene ring are more suitable for pathogenic bacteria with higher cholesterol content in their cell membranes.

[0037] Aggregatibacter actinomycetemcomitans, as one of the main pathogens of dental periodontitis, has a significantly higher cholesterol content in its cell membrane than other periodontal pathogens. Therefore, it can be inserted into the cell membrane by thiophene rings to affect the life activities of the pathogens, thereby enhancing the antibacterial performance of oral repair membranes against Aggregatibacter actinomycetemcomitans.

[0038] Subsequently, the present invention mixes the imidazole-thiophene intermediate with gelatin, and utilizes the carboxyl groups in the imidazole-thiophene intermediate to perform an amidation reaction with the amino groups in the gelatin, thereby introducing imidazole rings, thiophene rings and crosslinkable double bonds into the gelatin. In order to avoid self-crosslinking of double bonds during the amidation reaction with gelatin, the present invention also adds hydroquinone as a polymerization inhibitor in this process. Hydroquinone can interrupt the growth of the polymerization chain by capturing free radicals, and can inhibit the self-polymerization reaction of acrylate double bonds in the imidazole-thiophene intermediate, ensuring that the amidation grafting reaction proceeds preferentially, improving grafting efficiency and product structural uniformity. Then, the gelatin after the reaction is dialyzed using a dialysis bag to remove ungrafted monomers, and finally obtains antibacterial crosslinkable gelatin with antibacterial properties.

[0039] Based on this, the antibacterial crosslinkable gelatin prepared in this invention is mixed with GelMA gelatin. Hydroxyapatite is also added to the mixed solution as a bone-inducing material. Then, under the action of a photoinitiator, an antibacterial hydrogel material is finally formed. This material is then bonded to an absorbable PLGA membrane to obtain an oral repair membrane material. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the preparation process of the oral repair membrane material of the present invention;

[0041] Figure 2 This is the chemical reaction equation for step a of the present invention;

[0042] Figure 3 This is the chemical reaction equation for step b of the present invention;

[0043] Figure 4 This is the chemical reaction equation for step c of the present invention. Detailed Implementation

[0044] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] The GelMA gelatin used in the embodiments and comparative examples of this invention is commercially available type M752860 methacrylamide gelatin; the photoinitiator used is Irgacure 2959 photoinitiator; the hydroxyapatite used is commercially available type HAP00200 nano hydroxyapatite; the gelatin used is type G108395 gelatin; the Porphyromonas gingivalis used is commercially available type BNCC353909 Porphyromonas gingivalis; and the Aggregatibacter actinomycetemcomitans used is commercially available type BNCC337071 Aggregatibacter actinomycetemcomitans.

[0046] Example 1

[0047] A method for preparing an oral repair membrane material includes the following steps:

[0048] S1. Antibacterial crosslinkable modified gelatin and methacrylamide gelatin were mixed and added to PBS buffer to prepare a gelatin solution with a concentration of 10wt%. The solution was heated to 50℃ and stirred until completely dissolved. Hydroxyapatite and photoinitiator were added to the solution and ultrasonically stirred until homogeneous. The solution was then poured into a molding container with a depth of 0.45mm and irradiated with ultraviolet light for 60s to form an antibacterial hydrogel.

[0049] The mass ratio of the antibacterial crosslinkable modified gelatin, methacrylamide gelatin, hydroxyapatite and photoinitiator is 1:9:3:0.15.

[0050] The preparation method of the antibacterial crosslinkable modified gelatin includes the following steps:

[0051] a. Dissolve methacrylic acid in anhydrous dichloromethane, disperse and mix evenly, add thionyl chloride and mix evenly, add DMF, under nitrogen atmosphere protection, heat to 40°C, reflux and stir for 6 hours, remove excess solvent by rotary evaporation to obtain methacryloyl chloride intermediate;

[0052] The mass ratio of methacrylic acid, sulfoxide, and DMF is 1:1.3:0.05;

[0053] b. Dissolve 1H-imidazol-4,5-diethanol and triethylamine in anhydrous dichloromethane, mix thoroughly, cool to a constant temperature in an ice-water bath, and add dropwise an anhydrous dichloromethane solution containing the methacryloyl chloride intermediate prepared in step a), stirring continuously during the dropwise addition. After the dropwise addition is completed, heat the reaction system to 25°C and stir for 8 hours. Wash the resulting reaction solution three times with saturated sodium chloride solution and deionized water, collect the organic phase, dry it overnight with anhydrous sodium sulfate, filter, and vacuum dry the filtrate to obtain the imidazolium intermediate.

[0054] The mass ratio of the 1H-imidazol-4,5-diethanol, triethylamine, and methacryloyl chloride intermediate is 1:0.8:0.7.

[0055] c. Disperse the imidazole intermediate in anhydrous ethanol, stir until evenly dispersed, and set aside for later use;

[0056] 3-Thiophene malonic acid was added to deionized water, and NHS and EDC·HCl were added to it. After stirring and activating for 1 hour in the dark, an anhydrous ethanol solution of imidazole intermediate was added dropwise. After the addition was completed, the reaction was stirred at room temperature for 18 hours, and the pH of the reaction system was controlled at 6-7 during the stirring process. After the reaction was completed, hydrochloric acid was added dropwise to the reaction solution to adjust the pH of the solution to 2. After stirring for 1 hour, the precipitate was separated by filtration, washed 5 times with deionized water, and dried to constant weight to obtain imidazole-thiophene intermediate.

[0057] The mass ratio of the imidazole intermediate, 3-thiophene malonic acid, NHS, and EDC·HCl is 1:1:0.6:1.

[0058] d. Mix gelatin with PBS buffer, heat to 50°C in a water bath, stir until the gelatin is completely dissolved, cool to room temperature, add imidazole-thiophene intermediate, hydroquinone, EDC·HCl and NHS, heat to 40°C, stir for 4 hours, add 4 times the volume of PBS buffer deionized water to stop the reaction, dialyze the mixture for 5 days, freeze-dry the product in the dialysate bag to obtain antibacterial crosslinkable modified gelatin;

[0059] The mass ratio of gelatin, imidazole-thiophene intermediate, hydroquinone, EDC·HCl, and NHS is 1:0.5:0.04:2.5:1.7.

[0060] During dialysis, a 3kDa dialysis bag was used; the dialysis temperature was 4℃; and the dialysis fluid was changed every 6 hours.

[0061] S2. After attaching the antibacterial hydrogel to the inner side of the back membrane, cut it into shape, and dry it to obtain the oral repair membrane material.

[0062] Example 2

[0063] A method for preparing an oral repair membrane material, compared with Example 1, this example changes the amount of antibacterial crosslinkable gelatin and methacrylamide gelatin added in step S1, while the remaining steps remain unchanged, specifically including the following steps:

[0064] S1. Antibacterial crosslinkable modified gelatin and methacrylamide gelatin were mixed and added to PBS buffer to prepare a gelatin solution with a concentration of 10wt%. The solution was heated to 50℃ and stirred until completely dissolved. Hydroxyapatite and photoinitiator were added to the solution and ultrasonically stirred until homogeneous. The solution was then poured into a molding container with a depth of 0.45mm and irradiated with ultraviolet light for 60s to form an antibacterial hydrogel.

[0065] The mass ratio of the antibacterial crosslinkable modified gelatin, methacrylamide gelatin, hydroxyapatite and photoinitiator is 2:8:3:0.15.

[0066] S2. After attaching the antibacterial hydrogel to the inner side of the back membrane, cut it into shape, and dry it to obtain the oral repair membrane material.

[0067] Example 3

[0068] A method for preparing an oral repair membrane material, compared with Example 1, this example increases the amount of imidazole-thiophene intermediate added in step d, and correspondingly increases the amount of EDC·HCl and NHS added, while the remaining steps remain unchanged, specifically including the following steps:

[0069] a. Dissolve methacrylic acid in anhydrous dichloromethane, disperse and mix evenly, add thionyl chloride and mix evenly, add DMF, under nitrogen atmosphere protection, heat to 40°C, reflux and stir for 6 hours, remove excess solvent by rotary evaporation to obtain methacryloyl chloride intermediate;

[0070] The mass ratio of methacrylic acid, sulfoxide, and DMF is 1:1.3:0.05;

[0071] b. Dissolve 1H-imidazol-4,5-diethanol and triethylamine in anhydrous dichloromethane, mix thoroughly, cool to a constant temperature in an ice-water bath, and add dropwise an anhydrous dichloromethane solution containing the methacryloyl chloride intermediate prepared in step a), stirring continuously during the dropwise addition. After the dropwise addition is completed, heat the reaction system to 25°C and stir for 8 hours. Wash the resulting reaction solution three times with saturated sodium chloride solution and deionized water, collect the organic phase, dry it overnight with anhydrous sodium sulfate, filter, and vacuum dry the filtrate to obtain the imidazolium intermediate.

[0072] The mass ratio of the 1H-imidazol-4,5-diethanol, triethylamine, and methacryloyl chloride intermediate is 1:0.8:0.7.

[0073] c. Disperse the imidazole intermediate in anhydrous ethanol, stir until evenly dispersed, and set aside for later use;

[0074] 3-Thiophene malonic acid was added to deionized water, and NHS and EDC·HCl were added to it. After stirring and activating for 1 hour in the dark, an anhydrous ethanol solution of imidazole intermediate was added dropwise. After the addition was completed, the reaction was stirred at room temperature for 18 hours, and the pH of the reaction system was controlled at 6-7 during the stirring process. After the reaction was completed, hydrochloric acid was added dropwise to the reaction solution to adjust the pH of the solution to 2. After stirring for 1 hour, the precipitate was separated by filtration, washed 5 times with deionized water, and dried to constant weight to obtain imidazole-thiophene intermediate.

[0075] The mass ratio of the imidazole intermediate, 3-thiophene malonic acid, NHS, and EDC·HCl is 1:1:0.6:1.

[0076] d. Mix gelatin with PBS buffer, heat to 50°C in a water bath, stir until the gelatin is completely dissolved, cool to room temperature, add imidazole-thiophene intermediate, hydroquinone, EDC·HCl and NHS, heat to 40°C, stir for 4 hours, add 4 times the volume of PBS buffer deionized water to stop the reaction, dialyze the mixture for 5 days, freeze-dry the product in the dialysate bag to obtain antibacterial crosslinkable modified gelatin;

[0077] The mass ratio of gelatin, imidazole-thiophene intermediate, hydroquinone, EDC·HCl, and NHS is 1:0.9:0.04:2.5:1.7.

[0078] During dialysis, a 3kDa dialysis bag was used; the dialysis temperature was 4℃; and the dialysis fluid was changed every 6 hours.

[0079] Example 4

[0080] A method for preparing an oral repair membrane material, compared with Example 1, this example increases the amount of photoinitiator added in step S1, while the remaining steps remain unchanged, specifically including the following steps:

[0081] S1. Mix antibacterial crosslinkable modified gelatin with methacrylamide gelatin, add the mixture to PBS buffer to prepare a 10wt% gelatin solution, heat to 50℃, stir until completely dissolved, add hydroxyapatite and photoinitiator, sonicate and mix evenly, pour into a molding container with a depth of 0.45mm, irradiate with ultraviolet light for 60s to form, and obtain antibacterial hydrogel;

[0082] The mass ratio of the antibacterial crosslinkable modified gelatin, methacrylamide gelatin, hydroxyapatite and photoinitiator is 1:9:3:0.35.

[0083] S2. After attaching the antibacterial hydrogel to the inner side of the back membrane, cut it into shape, and dry it to obtain the oral repair membrane material.

[0084] Example 5

[0085] A method for preparing an oral repair membrane material includes the following steps:

[0086] S1. Antibacterial crosslinkable modified gelatin and methacrylamide gelatin were mixed and added to PBS buffer to prepare a gelatin solution with a concentration of 10wt%. The solution was heated to 50℃ and stirred until completely dissolved. Hydroxyapatite and photoinitiator were added to the solution and ultrasonically stirred until homogeneous. The solution was then poured into a molding container with a depth of 0.45mm and irradiated with ultraviolet light for 60s to form an antibacterial hydrogel.

[0087] The mass ratio of the antibacterial crosslinkable modified gelatin, methacrylamide gelatin, hydroxyapatite and photoinitiator is 1:9:5:0.15.

[0088] The preparation method of the antibacterial crosslinkable modified gelatin includes the following steps:

[0089] a. Dissolve methacrylic acid in anhydrous dichloromethane, disperse and mix evenly, add thionyl chloride and mix evenly, add DMF, under nitrogen atmosphere protection, heat to 40°C, reflux and stir for 6 hours, remove excess solvent by rotary evaporation to obtain methacryloyl chloride intermediate;

[0090] The mass ratio of methacrylic acid, sulfoxide, and DMF is 1:1.6:0.05;

[0091] b. Dissolve 1H-imidazol-4,5-diethanol and triethylamine in anhydrous dichloromethane, mix thoroughly, cool to a constant temperature in an ice-water bath, and add dropwise an anhydrous dichloromethane solution containing the methacryloyl chloride intermediate prepared in step a), stirring continuously during the dropwise addition. After the dropwise addition is completed, heat the reaction system to 25°C and stir for 8 hours. Wash the resulting reaction solution three times with saturated sodium chloride solution and deionized water, collect the organic phase, dry it overnight with anhydrous sodium sulfate, filter, and vacuum dry the filtrate to obtain the imidazolium intermediate.

[0092] The mass ratio of the 1H-imidazol-4,5-diethanol, triethylamine, and methacryloyl chloride intermediate is 1:1.3:0.9.

[0093] c. Disperse the imidazole intermediate in anhydrous ethanol, stir until evenly dispersed, and set aside for later use;

[0094] 3-Thiophene malonic acid was added to deionized water, and NHS and EDC·HCl were added to it. After stirring and activating for 1 hour in the dark, an anhydrous ethanol solution of imidazole intermediate was added dropwise. After the addition was completed, the reaction was stirred at room temperature for 18 hours, and the pH of the reaction system was controlled at 6-7 during the stirring process. After the reaction was completed, hydrochloric acid was added dropwise to the reaction solution to adjust the pH of the solution to 2. After stirring for 1 hour, the precipitate was separated by filtration, washed 5 times with deionized water, and dried to constant weight to obtain imidazole-thiophene intermediate.

[0095] The mass ratio of the imidazole intermediate, 3-thiophene malonic acid, NHS, and EDC·HCl is 1.2:1:0.65:1.1.

[0096] d. Mix gelatin with PBS buffer, heat to 50°C in a water bath, stir until the gelatin is completely dissolved, cool to room temperature, add imidazole-thiophene intermediate, hydroquinone, EDC·HCl and NHS, heat to 40°C, stir for 4 hours, add 4 times the volume of PBS buffer deionized water to stop the reaction, dialyze the mixture for 5 days, freeze-dry the product in the dialysate bag to obtain antibacterial crosslinkable modified gelatin;

[0097] The mass ratio of gelatin, imidazole-thiophene intermediate, hydroquinone, EDC·HCl, and NHS is 1:0.5:0.04:2.5:1.7.

[0098] During dialysis, a 3kDa dialysis bag was used; the dialysis temperature was 4℃; and the dialysis fluid was changed every 6 hours.

[0099] S2. After attaching the antibacterial hydrogel to the inner side of the back membrane, cut it into shape, and dry it to obtain the oral repair membrane material.

[0100] Comparative Example 1

[0101] A method for preparing an oral repair membrane material, compared with Example 1, in step S1 of this comparative example, no antibacterial crosslinkable modified gelatin was prepared or added, and the other operation steps and process parameters were exactly the same as in Example 1;

[0102] Specifically, the following steps are included:

[0103] S1. Methacrylamide gelatin was added to PBS buffer to prepare a gelatin solution with a concentration of 10wt%. The solution was heated to 50℃ and stirred until completely dissolved. Hydroxyapatite and photoinitiator were added to the solution. After ultrasonic stirring and mixing, the solution was poured into a molding container with a depth of 0.45mm. After molding by irradiating with ultraviolet light for 60s, an antibacterial hydrogel was obtained.

[0104] The mass ratio of the methacrylamide gelatin, hydroxyapatite and photoinitiator is 10:3:0.15.

[0105] S2. After bonding the hydrogel to the inner side of the back membrane, cut it into shape, and dry it to obtain the oral repair membrane material.

[0106] Comparative Example 2

[0107] A method for preparing an oral repair membrane material, compared with Example 1, this comparative example did not prepare a methacryloyl chloride intermediate, but directly used methacrylic acid for step b treatment, and the other operation steps and process parameters were exactly the same as those in Example 1;

[0108] The preparation method of the antibacterial crosslinkable modified gelatin includes the following steps:

[0109] a. Dissolve 1H-imidazol-4,5-diethanol and triethylamine in anhydrous dichloromethane, mix thoroughly, cool to a constant temperature in an ice-water bath, and add anhydrous dichloromethane solution containing methacrylic acid dropwise while stirring continuously. After the addition is complete, heat the reaction system to 25°C and stir for 8 hours. Wash the resulting reaction solution three times with saturated sodium chloride solution and deionized water, collect the organic phase, dry it overnight with anhydrous sodium sulfate, filter, and vacuum dry the filtrate to obtain the imidazolium intermediate.

[0110] The mass ratio of 1H-imidazol-4,5-diethanol, triethylamine, and methacrylic acid is 1:0.8:0.7.

[0111] b. Disperse the imidazole intermediate in anhydrous ethanol, stir until evenly dispersed, and set aside for later use;

[0112] 3-Thiophene malonic acid was added to deionized water, and NHS and EDC·HCl were added to it. After stirring and activating for 1 hour in the dark, an anhydrous ethanol solution of imidazole intermediate was added dropwise. After the addition was completed, the reaction was stirred at room temperature for 18 hours, and the pH of the reaction system was controlled at 6-7 during the stirring process. After the reaction was completed, hydrochloric acid was added dropwise to the reaction solution to adjust the pH of the solution to 2. After stirring for 1 hour, the precipitate was separated by filtration, washed 5 times with deionized water, and dried to constant weight to obtain imidazole-thiophene intermediate.

[0113] The mass ratio of the imidazole intermediate, 3-thiophene malonic acid, NHS, and EDC·HCl is 1:1:0.6:1.

[0114] c. Mix gelatin with PBS buffer, heat to 50°C in a water bath, stir until the gelatin is completely dissolved, cool to room temperature, add imidazole-thiophene intermediate, hydroquinone, EDC·HCl and NHS, heat to 40°C, stir for 4 hours, add 4 times the volume of PBS buffer deionized water to stop the reaction, dialyze the mixture for 5 days, freeze-dry the product in the dialysis bag to obtain antibacterial crosslinkable modified gelatin;

[0115] The mass ratio of gelatin, imidazole-thiophene intermediate, hydroquinone, EDC·HCl, and NHS is 1:0.5:0.04:2.5:1.7.

[0116] During dialysis, a 3kDa dialysis bag was used; the dialysis temperature was 4℃; and the dialysis fluid was changed every 6 hours.

[0117] Comparative Example 3

[0118] A method for preparing an oral repair membrane material, compared with Example 1, this comparative example uses isobutyric acid to replace methacrylic acid in an equal amount, and the other steps remain unchanged;

[0119] The preparation method of the antibacterial crosslinkable modified gelatin includes the following steps:

[0120] a. Dissolve isobutyric acid in anhydrous dichloromethane, disperse and mix evenly, add thionyl chloride and mix evenly, add DMF, under nitrogen atmosphere protection, heat to 40°C, reflux and stir for 6 hours, remove excess solvent by rotary evaporation to obtain isobutyryl chloride intermediate;

[0121] The mass ratio of isobutyric acid, thionyl chloride, and DMF is 1:1.3:0.05;

[0122] b. Dissolve 1H-imidazol-4,5-diethanol and triethylamine in anhydrous dichloromethane, mix thoroughly, cool to a constant temperature in an ice-water bath, and add dropwise an anhydrous dichloromethane solution containing the isobutyryl chloride intermediate prepared in step a), stirring continuously during the dropwise addition. After the dropwise addition is completed, heat the reaction system to 25°C and stir for 8 hours. Wash the resulting reaction solution three times with saturated sodium chloride solution and deionized water, collect the organic phase, dry it overnight with anhydrous sodium sulfate, filter, and vacuum dry the filtrate to obtain the imidazolium intermediate.

[0123] The mass ratio of the intermediate 1H-imidazol-4,5-diethanol, triethylamine, and isobutyryl chloride is 1:0.8:0.7.

[0124] c. Disperse the imidazole intermediate in anhydrous ethanol, stir until evenly dispersed, and set aside for later use;

[0125] 3-Thiophene malonic acid was added to deionized water, and NHS and EDC·HCl were added to it. After stirring and activating for 1 hour in the dark, an anhydrous ethanol solution of imidazole intermediate was added dropwise. After the addition was completed, the reaction was stirred at room temperature for 18 hours, and the pH of the reaction system was controlled at 6-7 during the stirring process. After the reaction was completed, hydrochloric acid was added dropwise to the reaction solution to adjust the pH of the solution to 2. After stirring for 1 hour, the precipitate was separated by filtration, washed 5 times with deionized water, and dried to constant weight to obtain imidazole-thiophene intermediate.

[0126] The mass ratio of the imidazole intermediate, 3-thiophene malonic acid, NHS, and EDC·HCl is 1:1:0.6:1.

[0127] d. Mix gelatin with PBS buffer, heat to 50°C in a water bath, stir until the gelatin is completely dissolved, cool to room temperature, add imidazole-thiophene intermediate, hydroquinone, EDC·HCl and NHS, heat to 40°C, stir for 4 hours, add 4 times the volume of PBS buffer deionized water to stop the reaction, dialyze the mixture for 5 days, freeze-dry the product in the dialysate bag to obtain antibacterial crosslinkable modified gelatin;

[0128] The mass ratio of gelatin, imidazole-thiophene intermediate, hydroquinone, EDC·HCl, and NHS is 1:0.5:0.04:2.5:1.7.

[0129] During dialysis, a 3kDa dialysis bag was used; the dialysis temperature was 4℃; and the dialysis fluid was changed every 6 hours.

[0130] The performance of the above embodiments and comparative examples was tested, as follows:

[0131] Cytotoxicity test: Cytotoxicity test was performed according to GB / T 16886.5-2017. Grade 0 is no cytotoxicity, Grade 1 is slight cytotoxicity, Grade 2 is moderate cytotoxicity, and Grade 3 is severe cytotoxicity.

[0132] Antibacterial performance testing: *Porphyromonas gingivalis* and *Actinomyces actinomycetes* were inoculated and cultured separately into CDC anaerobic blood agar plates. After anaerobic culture to the third generation, they were inoculated into BHI medium and cultured anaerobically at 37°C for 48 hours. The resulting cultures were then prepared to a bacterial concentration of 1×10⁻⁶. 6 The bacterial suspension of cfu / mL was inoculated onto the antibacterial hydrogel surface of the oral repair membrane material prepared in Examples 1-5 and Comparative Examples 1-3. After culturing at 37°C for 12 hours, 5 mL of BHI liquid culture medium and 45 mL of sterile physiological saline were mixed to prepare an rinsing solution. The solutions were rinsed separately, and the eluent was collected to obtain bacterial suspensions for detecting Porphyromonas gingivalis and Actinobacillus actinomycetii.

[0133] The obtained bacterial suspensions were inoculated into new BHI agar plates and anaerobic cultured at 37°C for 12 hours. The plates were then counted, and their antibacterial rate was calculated.

[0134] ;

[0135] Anti-degradation performance test: The antibacterial hydrogels prepared in Examples 1-5 and Comparative Examples 1-3 were dried and weighed. Then, they were immersed in SBF solution at 37°C. On days 7, 28, and 56, the gels were filtered out, dried, and the weight difference was calculated to obtain the degradation rate. The test results are shown in Table 1 below.

[0136] Table 1. Performance test results of the antibacterial hydrogels prepared in Examples 1-5 and Comparative Examples 1-3

[0137]

[0138] A comparison of the data from Examples 1 and 2 of this invention shows that the antibacterial activity against periodontal pathogens was significantly improved after the addition of antibacterial crosslinkable gelatin. However, the degradation rate also increased to some extent. This is because the crosslinking performance of the antibacterial crosslinkable gelatin prepared in this invention is given by the imidazole-thiophene intermediate. Compared with conventional gelatin modified with methacrylic anhydride, the imidazole-thiophene intermediate has greater steric hindrance. During crosslinking with the photoinitiator, it can lead to insufficient crosslinking, thus causing an increase in the degradation rate.

[0139] As can be seen from the comparison between Example 1 and Example 3 of the present invention, after increasing the amount of imidazole-thiophene intermediate added, its antibacterial and anti-degradation properties were improved to a certain extent. The reason is that increasing the amount of imidazole-thiophene intermediate increased the success rate of grafting imidazole-thiophene intermediate onto gelatin in step d, and increased the content of imidazole, thiophene and crosslinkable double bonds in gelatin.

[0140] As can be seen from the data of Examples 1 and 4, the addition of photoinitiator effectively improved the hydrogel's resistance to degradation. However, the increased photoinitiator content led to photoinitiator residue, causing slight cytotoxicity.

[0141] Comparing Example 1 and Example 5, it can be found that after increasing the amount of each component added in steps a to c, the reaction proceeds more thoroughly and the antibacterial rate of the product is effectively improved.

[0142] By comparing the data in Example 1, it can be found that the antibacterial properties of the hydrogel undoubtedly decreased significantly when the antibacterial crosslinkable gelatin was not prepared. However, since the antibacterial crosslinkable gelatin prepared in this invention was not used, the crosslinking caused by the photoinitiator was more thorough when preparing the hydrogel, which made the hydrogel have better anti-degradation ability.

[0143] Comparative Example 2 data shows that without acyl chlorination of methacrylic acid, subsequent processing steps lead to reduced reactivity and incomplete reaction, resulting in a decrease in double bonds in the final synthesized antibacterial crosslinkable gelatin, thus significantly reducing the hydrogel's resistance to degradation. However, since the introduction of thiophene and imidazole rings in subsequent steps does not involve double bonds, the gelatin still retains some antibacterial properties. However, because the reaction between methacrylic acid and 1H-imidazol-4,5-diethanol in step b is incomplete, some unreacted 1H-imidazol-4,5-diethanol participates in the reaction of 3-thiophene malonic acid in subsequent steps, ultimately forming disordered esters. This affects the purity of the final synthesized thiophene-imidazol intermediate, leading to a decrease in the success rate of grafting the thiophene-imidazol intermediate onto the gelatin, thus resulting in a certain degree of decrease in antibacterial properties.

[0144] The data from Comparative Example 3 proves that the double bonds in the antibacterial crosslinkable modified gelatin prepared in this invention originate from methacrylic acid.

[0145] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an oral repair membrane material, characterized in that, Includes the following steps: S1. Mix antibacterial crosslinkable modified gelatin with methacrylamide gelatin, add the mixture to PBS buffer, heat and stir until completely dissolved, add hydroxyapatite and photoinitiator, sonicate and mix evenly, add to molding container, use ultraviolet light to mold, and obtain antibacterial hydrogel. The preparation method of the antibacterial crosslinkable modified gelatin specifically includes the following steps: a. Dissolve methacrylic acid in anhydrous dichloromethane, disperse and mix evenly, add thionyl chloride and mix evenly, add DMF, under nitrogen atmosphere protection, heat and reflux and stir for 4-6 hours, remove excess solvent by rotary evaporation to obtain methacryloyl chloride intermediate; b. Dissolve 1H-imidazol-4,5-diethanol and triethylamine in anhydrous dichloromethane, mix thoroughly, cool to a constant temperature in an ice-water bath, and add dropwise an anhydrous dichloromethane solution containing the methacryloyl chloride intermediate prepared in step a), stirring continuously during the dropwise addition. After the dropwise addition is completed, heat the reaction system and stir the reaction. Wash the resulting reaction solution 2-3 times with saturated sodium chloride solution and deionized water, collect the organic phase, dry it overnight with anhydrous sodium sulfate, filter, and vacuum dry the filtrate to obtain the imidazolium intermediate; c. Disperse the imidazole intermediate in anhydrous ethanol, and stir until the dispersion is uniform to obtain an anhydrous ethanol solution containing the imidazole intermediate; 3-Thiophene malonic acid was added to deionized water, and NHS and EDC·HCl were added to it. After activation by stirring in the dark, an anhydrous ethanol solution containing the imidazole intermediate was added dropwise. After the addition was completed, the reaction was stirred at room temperature for 12-18 hours, and the pH of the reaction system was controlled at 6-7 during the stirring process. After the reaction was completed, hydrochloric acid was added dropwise to the reaction solution to adjust the pH of the solution to 1.5-2. After stirring for 0.5-1 hour, the precipitate was separated by filtration, washed with deionized water 3-5 times, and dried to obtain the imidazole-thiophene intermediate. d. Mix gelatin with PBS buffer and stir until the gelatin is completely dissolved. Cool to room temperature, add imidazole-thiophene intermediate, hydroquinone, EDC·HCl and NHS. Heat and stir to react. Add 3-4 times the volume of PBS buffer and deionized water to stop the reaction. Dialyze the mixture for 3-5 days and freeze-dry the product in the dialysis bag to obtain antibacterial crosslinkable modified gelatin. S2. After attaching the antibacterial hydrogel to the inner side of the back membrane, cut it into shape, and dry it to obtain the oral repair membrane material.

2. The method for preparing an oral repair membrane material according to claim 1, characterized in that: In step a, the mass ratio of methacrylic acid, sulfoxide, and DMF is 1:(1.3~1.6):(0.05~0.1). When heating, the heating temperature is 38~40℃.

3. The method for preparing an oral repair membrane material according to claim 1, characterized in that: In step b, the mass ratio of 1H-imidazol-4,5-diethanol, triethylamine, and methacryloyl chloride intermediate is 1:(0.8~1.3):(0.7~0.9). When the reaction system is heated and stirred, the heating temperature is 24~26℃ and the reaction time is 6~8h.

4. The method for preparing an oral repair membrane material according to claim 1, characterized in that: In step c, the mass ratio of 3-thiophene malonic acid, NHS, EDC·HCl, and imidazole intermediate is 1:(0.6~0.65):(1~1.1):(1~1.2).

5. The method for preparing an oral repair membrane material according to claim 1, characterized in that: In step d, the mass ratio of gelatin, imidazole-thiophene intermediate, hydroquinone, EDC·HCl and NHS is 1:(0.5~0.9):(0.01~0.04):(2.5~4.5):(1.7~3.5). Heat to 38~45℃, and stir the reaction. The heating temperature is 38~45℃, and the reaction time is 3~4h.

6. The method for preparing an oral repair membrane material according to claim 1, characterized in that: In step d, the molecular weight cutoff of the dialysis bag used during dialysis is 3-5 kDa; the dialysis temperature is 2-8℃, and the dialysis solution is changed every 6-8 hours.

7. The method for preparing an oral repair membrane material according to claim 1, characterized in that: In step S1, the mass ratio of antibacterial crosslinkable modified gelatin, methacrylamide gelatin, hydroxyapatite, and photoinitiator is (1~2):(8~9):(3~5):(0.15~0.35).

8. The method for preparing an oral repair membrane material according to claim 1, characterized in that: In step S1, the temperature is raised to 48~52℃ and irradiated with ultraviolet light for 65±5s; in step S2, the back film is a PLGA film.

9. An oral repair membrane material prepared by the preparation method according to any one of claims 1 to 8.

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