Preparation method of antibacterial periosteum for oral cavity

Abstract

The invention discloses a preparation method of an oral antibacterial periosteum, and relates to the technical field of periosteum. Comprising the following steps: step 1, preparing raw materials; 2, pig peritoneum pretreatment: removing large-area fat from the pig peritoneum, trimming miscellaneous edges, and cleaning with pure water until no obvious attachments exist, so as to obtain a pig peritoneum A; step 3, degreasing treatment: putting the pig peritoneum A into a sodium hydroxide solution for degreasing cleaning, and then sequentially performing pure water cleaning and isopropanol cleaning to obtain a pig peritoneum B; 4, decellularization treatment: putting the pig peritoneum B into a sodium chloride solution for decellularization cleaning, and then cleaning with pure water to obtain a pig peritoneum C; step 5, cleaning: further cleaning the pig peritoneum C with pure water, and then performing ultrasonic cleaning to obtain pig peritoneum D; 6, the pig peritoneum D is flatly laid and freeze-dried, and a basic periosteum is obtained; and step 7, uniformly coating the surface of the basic periosteum with the carboxymethyl chitosan solution, spreading, and freeze-drying to obtain the antibacterial periosteum.

Description

Technical Field

[0001] The invention relates to the technical field of periosteum, in particular to a method for preparing an antibacterial periosteum for oral cavity. Background Art

[0002] The advantage of oral periosteum in the field of oral implants is that it can be placed between oral soft tissue and bone defects, thereby establishing a biological barrier, helping to maintain the position and shape of bone powder to prevent its loss, and preventing soft tissue from growing into the bone graft area while promoting the combination of bone powder and surrounding bone tissue, thereby improving the stability of the implant. Therefore, in the case of insufficient alveolar bone or abnormal morphology, implanting periosteum is a necessary step to ensure the long-term stability and functional recovery of dental implants.

[0003] However, bacterial infection is inevitable during surgery or postoperative recovery. About one million people suffer from medical device infection every year, which greatly increases the pain of patients and the cost of treatment. Carboxymethyl chitosan is a water-soluble chitosan derivative with many medical effects, such as promoting wound healing, analgesia and antibacterial effects. At the same time, carboxymethyl chitosan has good biocompatibility and biodegradability. Its aqueous solution has high viscosity and can effectively adhere to the periosteum surface. Therefore, carboxymethyl chitosan can be used as an effective coating for antibacterial periosteum.

[0004] In summary, in order to solve the above problems, an oral antibacterial periosteum is prepared, which makes the periosteum have good anti-bacterial infection and biocompatibility during use, and has important value in clinical applications. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing an oral antibacterial periosteum to solve the problems raised in the above background technology.

[0006] In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing an oral antibacterial periosteum, characterized in that: Step 1: Prepare raw materials; Step 2: Pretreatment of pig peritoneum: remove large areas of fat from the pig peritoneum, trim the edges, and wash with pure water until there is no obvious attachment to obtain pig peritoneum A; Step 3: Degreasing treatment: placing the porcine peritoneum A in a sodium hydroxide solution for degreasing and washing, and then washing with pure water and isopropanol in sequence to obtain the porcine peritoneum B; Step 4: decellularization treatment: placing the porcine peritoneum B in a sodium chloride solution for decellularization and washing, and then washing with pure water to obtain porcine peritoneum C; Step 5: Cleaning: The porcine peritoneum C is further cleaned with pure water and then ultrasonically cleaned to obtain porcine peritoneum D; Step 6: Spread the porcine peritoneum D on a freeze-drying plate and freeze-dry it to obtain the basic periosteum; Step 7: (1) Add carboxymethyl chitosan to pure water and stir evenly to obtain a carboxymethyl chitosan solution with a concentration of 1-5 wt%; (2) Evenly coat the carboxymethyl chitosan solution on the surface of the base periosteum, spread it on a freeze-drying plate, and freeze-dry to obtain an antibacterial periosteum.

[0007] More optimally, in step 3, during the degreasing and cleaning process, the temperature is 28-32°C, the shaking speed is 130-140 rpm, the time is 3-5 hours, and the number of degreasing and cleaning times is 2-3 times; During the pure water washing process, the temperature is 28-32°C, the shaking speed is 130-140 rpm, the time is 15-25 minutes, and the number of pure water washings is 4-6 times; During the isopropanol cleaning process, the temperature is 38-42° C., the shaking speed is 130-140 rpm, the time is 7-9 hours, and the number of isopropanol cleanings is 2-3 times.

[0008] More optimally, in step 4, during the decellularization washing process, the temperature is room temperature, the shaking speed is 130-140 rpm, and the time is 1-1.5 hours; during the pure water washing process, the temperature is room temperature, the shaking speed is 130-140 rpm, and the time is 0.5-0.6 hours; In step 5, during the pure water cleaning process, the temperature is room temperature, the shaking speed is 130-140 rpm, the time is 0.5-0.6 hours, and the number of pure water cleanings is 3-5 times; during the ultrasonic cleaning process, the temperature is room temperature, the time is 15-20 minutes, and the number of ultrasonic cleanings is 2-3 times.

[0009] More optimally, in step 3, the concentration of the sodium hydroxide solution is 0.01M~0.1M.

[0010] More optimally, in step 4, the concentration of the sodium chloride solution is 0.5M~4M.

[0011] Preferably, in step 7, the coating amount of the carboxymethyl chitosan solution is 0.05-0.5 g / cm 2 .

[0012] In a further embodiment, the steps in step 7 are as follows: (1) adding 1.5-2.5 wt% of carboxymethyl chitosan to 1-2 v / v% of acetic acid solution and stirring evenly to obtain solution A; adding 0.2-0.4 wt% of aldehyded hyaluronic acid and 0.6-0.8 wt% of quaternized hyaluronic acid to pure water and stirring evenly to obtain solution B; mixing equal volumes of solution A and solution B evenly to obtain a coating solution; (2) adding 0.05-0.1 wt% hydrogen peroxide and 0.002-0.004 wt% laccase into pure water to obtain an interfacial layer solution; (3) Pre-spray the interface layer solution on the surface of the base periosteum at a spraying amount of 0.02-0.03 g / cm2; then evenly coat the coating solution on the surface at a coating amount of 0.25-0.35 g / cm2; spread it on a freeze-drying plate and let it stand at room temperature for 1-2 hours; then pre-freeze it at -20°C-25°C for 4-6 hours, and freeze-dry it at -50°C-60°C for 24-48 hours to obtain the antibacterial periosteum; More optimally, the preparation method of the aldehyde-modified hyaluronic acid is as follows: adding low molecular weight hyaluronic acid to pure water and stirring evenly, adding sodium periodate solution dropwise, stirring for 4 to 6 hours in a dark environment, adding ethylene glycol for neutralization, dialyzing, and drying to obtain modified hyaluronic acid; under a nitrogen atmosphere, further adding it to pure water and stirring evenly, adding EDC and NHS and stirring evenly, adding dopamine, stirring at room temperature for 3 to 4 hours, dialyzing, and drying to obtain aldehyde-modified hyaluronic acid; The preparation method of the quaternized hyaluronic acid is as follows: adding a medium molecular weight hyaluronic acid to a 50-60wt% formamide aqueous solution and stirring evenly, heating to 60-65° C., adding a sodium hydroxide solution and 2,3-epoxypropyltrimethylammonium chloride, stirring for 3-4 hours, purifying, and drying to obtain the quaternized hyaluronic acid; More optimally, the average molecular weight of the hyaluronic acid is 8-10 kDa; the average molecular weight of the medium molecular hyaluronic acid is 100-150 kDa.

[0013] Compared with the prior art, the beneficial effects achieved by the present invention are: (1) The present invention provides a convenient and low-cost method for preparing an antibacterial periosteum, which effectively solves the problem of bacterial infection during implantation; and the prepared antibacterial periosteum has good biocompatibility and biodegradability.

[0014] (2) The antibacterial periosteum prepared by using carboxymethyl chitosan alone as a porous surface coating has the following advantages: first, the unique biological properties of carboxymethyl chitosan endow the periosteum with excellent bactericidal properties and good biocompatibility; second, the carboxymethyl chitosan coating has strong adhesion after infiltration and can fit closely to the implant area; third, the preparation of the carboxymethyl chitosan coating is simple and convenient, which can effectively reduce the production cost.

[0015] (3) In a further scheme of the present application, an antibacterial periosteum is prepared, and the mechanical properties and biocompatibility of the surface layer are effectively improved by introducing two hyaluronic acids with different molecular weights and different modifications into the coating.

[0016] Among them, the surface layer formed by a single carboxymethyl chitosan is brittle, so the introduction of hyaluronic acid forms a stable network through hydrogen bonding, van der Waals force, electrostatics, etc., to improve the toughness of the membrane. At the same time, the scheme uses the aldehyde group of the aldehyde-formylated hyaluronic acid and the catechol structure of the quaternized hyaluronic acid in two hyaluronic acid derivatives to achieve multi-network cross-linking, thereby generating toughness and improving the mechanical properties of the surface layer.

[0017] On the other hand, grafting catechol structure on low molecular weight hyaluronic acid can effectively improve bioadhesion and increase compatibility. At the same time, although the mechanical properties are improved by increasing the degree of cross-linking, the porous structure of the surface layer is still guaranteed; the reason is that the catechol structure will have overlapping electron clouds during the cross-linking process, and the phenolic hydroxyl group and the amino group in chitosan are prone to charge repulsion, thereby ensuring the porous structure. Quaternary ammonium salt hyaluronic acid has anti-inflammatory effects and can promote cell proliferation. In this way, the osteoinductivity of low molecular weight hyaluronic acid and the anti-inflammatory property of medium molecular weight hyaluronic acid are utilized to synergistically improve the biocompatibility of the antibacterial periosteum. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 is a scanning electron microscope image of the periosteum prepared in Comparative Example 1; Figure 2 is a scanning electron microscope image of the antibacterial periosteum prepared in Example 1; Figure 3 is a scanning electron microscopic image of the antibacterial periosteum prepared in Example 2; Figure 4 is a scanning electron microscopic image of the antibacterial periosteum prepared in Example 3; Figure 5 is a scanning electron microscopic image of the antibacterial periosteum prepared in Example 4; Figure 6 is a scanning electron microscopic image of the antibacterial periosteum prepared in Example 5; Figure 7 It is a graph showing the sterilization efficiency of the periosteum and antibacterial periosteum prepared in Comparative Example 1 and Examples 1-5; Figure 8 is a biocompatibility graph of the antibacterial periosteum prepared in Example 1; Fig. 9 is a biocompatibility graph of the antibacterial periosteum prepared in Example 2; Fig.10 is a biocompatibility graph of the antibacterial periosteum prepared in Example 3; Fig.11 is a biocompatibility graph of the antibacterial periosteum prepared in Example 4; Fig.12is a biocompatibility graph of the antibacterial periosteum prepared in Example 5; Fig.13 is a biocompatibility graph of the antibacterial periosteum prepared in Example 6; Fig.14 This is a graph showing the biocompatibility of the antibacterial periosteum prepared in Comparative Example 2; Fig.15 This is a macroscopic photograph of the antibacterial periosteum in Example 5. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Comparative Example 1: A method for preparing an oral antibacterial periosteum, comprising the following steps: Step 1: Raw material preparation: pig film; Step 2: Pretreatment of pig peritoneum: remove large areas of fat from the pig peritoneum, trim the edges, and rinse with running pure water until there is no obvious blood stain or other attachments on the surface of the pig peritoneum, thereby obtaining pig peritoneum A; Step 3: Degreasing treatment: Use 0.07M sodium hydroxide solution to degrease and clean the membrane A in a shaker (parameters: 30°C, 135rpm, 4 hours, 2 times); then wash with pure water (parameters: 30°C, 135rpm, 20min, 5 times); then wash with isopropanol (parameters: 40°C, 135rpm, 8h); obtain porcine peritoneum B; Step 4: Decellularization: Use 2M sodium chloride solution to decellularize and wash porcine peritoneum B in a shaker (parameters: room temperature, 135 rpm, 1 h); then wash with pure water (parameters: room temperature, 135 rpm, 0.5 h); obtain porcine peritoneum C; Step 5: Cleaning: The porcine peritoneum C is further cleaned with pure water (parameters: room temperature, 135 rpm, 0.5 h, 4 times), and then with ultrasound (parameters: room temperature, 20 min, 2 times) to obtain porcine peritoneum D; Step 6: Spread the cleaned pig peritoneum D on a freeze-drying plate, making sure that the pig peritoneum is as flat as possible, and freeze-dry it at -20°C for 24 hours to obtain the periosteum.

[0021] Embodiment 1: A method for preparing an oral antibacterial periosteum, comprising the following steps: Step 1: Raw material preparation: pig film, carboxymethyl chitosan; Step 2: Pretreatment of pig peritoneum: remove large areas of fat from the pig peritoneum, trim the edges, and rinse with running pure water until there is no obvious blood stain or other attachments on the surface of the pig peritoneum, thereby obtaining pig peritoneum A; Step 3: Degreasing treatment: Use 0.07M sodium hydroxide solution to degrease and clean the membrane A in a shaker (parameters: 30°C, 135rpm, 4 hours, 2 times); then wash with pure water (parameters: 30°C, 135rpm, 20min, 5 times); then wash with isopropanol (parameters: 40°C, 135rpm, 8h); obtain porcine peritoneum B; Step 4: Decellularization: Use 2M sodium chloride solution to decellularize and wash porcine peritoneum B in a shaker (parameters: room temperature, 135 rpm, 1 h); then wash with pure water (parameters: room temperature, 135 rpm, 0.5 h); obtain porcine peritoneum C; Step 5: Cleaning: The porcine peritoneum C is further cleaned with pure water (parameters: room temperature, 135 rpm, 0.5 h, 4 times), and then with ultrasound (parameters: room temperature, 20 min, 2 times) to obtain porcine peritoneum D; Step 6: Spread the cleaned porcine peritoneum D on a freeze-drying plate, make sure the porcine peritoneum is as flat as possible, and freeze-dry it at -20°C for 24 hours to obtain the basic periosteum; Step 7: (1) Add 1 g of carboxymethyl chitosan to 100 mL of pure water and heat and stir to dissolve to obtain a carboxymethyl chitosan solution; (2) Apply the carboxymethyl chitosan solution evenly on the surface of the base periosteum with a coating amount of 0.3 g / cm 2 ; Spread it flat on a freeze-drying plate, keep the pig peritoneum as flat as possible, freeze-dry it at -20℃ for 24h to obtain the antibacterial periosteum.

[0022] Embodiment 2: A method for preparing an oral antibacterial periosteum, comprising the following steps: Step 1: Raw material preparation: pig film, carboxymethyl chitosan; Step 2: Pretreatment of pig peritoneum: remove large areas of fat from the pig peritoneum, trim the edges, and rinse with running pure water until there is no obvious blood stain or other attachments on the surface of the pig peritoneum, thereby obtaining pig peritoneum A; Step 3: Degreasing treatment: Use 0.03M sodium hydroxide solution to degrease and clean the membrane A in a shaker (parameters: 30°C, 135rpm, 4 hours, 2 times); then clean it with pure water (parameters: 30°C, 135rpm, 20min, 5 times); then clean it with isopropanol (parameters: 40°C, 135rpm, 8h); obtain porcine peritoneum B; Step 4: Decellularization: Use 1M sodium chloride solution to decellularize and wash the porcine peritoneum B in a shaker (parameters: room temperature, 135 rpm, 1 h); then wash with pure water (parameters: room temperature, 135 rpm, 0.5 h); obtain porcine peritoneum C; Step 5: Cleaning: The porcine peritoneum C is further cleaned with pure water (parameters: room temperature, 135 rpm, 0.5 h, 4 times), and then with ultrasound (parameters: room temperature, 20 min, 2 times) to obtain porcine peritoneum D; Step 6: Spread the cleaned porcine peritoneum D on a freeze-drying plate, make sure the porcine peritoneum is as flat as possible, and freeze-dry it at -20°C for 24 hours to obtain the basic periosteum; Step 7: (1) Add 2 g of carboxymethyl chitosan to 100 mL of pure water and heat and stir to dissolve to obtain a carboxymethyl chitosan solution; (2) Apply the carboxymethyl chitosan solution evenly on the surface of the base periosteum with a coating amount of 0.1 g / cm 2 ; Spread it flat on a freeze-drying plate, keep the pig peritoneum as flat as possible, freeze-dry it at -20℃ for 24h to obtain the antibacterial periosteum.

[0023] Embodiment 3: A method for preparing an oral antibacterial periosteum, comprising the following steps: Step 1: Raw material preparation: pig film, carboxymethyl chitosan; Step 2: Pretreatment of pig peritoneum: remove large areas of fat from the pig peritoneum, trim the edges, and rinse with running pure water until there is no obvious blood stain or other attachments on the surface of the pig peritoneum, thereby obtaining pig peritoneum A; Step 3: Degreasing treatment: Use 0.08M sodium hydroxide solution to degrease and clean the film A in a shaker (parameters: 30°C, 135rpm, 4 hours, 2 times); then wash with pure water (parameters: 30°C, 135rpm, 20min, 5 times); then wash with isopropanol (parameters: 40°C, 135rpm, 8h); obtain porcine peritoneum B; Step 4: Decellularization: Use 2.5M sodium chloride solution to decellularize and wash the porcine peritoneum B in a shaker (parameters: room temperature, 135 rpm, 1 h); then wash with pure water (parameters: room temperature, 135 rpm, 0.5 h); obtain porcine peritoneum C; Step 5: Cleaning: The porcine peritoneum C is further cleaned with pure water (parameters: room temperature, 135 rpm, 0.5 h, 4 times), and then with ultrasound (parameters: room temperature, 20 min, 2 times) to obtain porcine peritoneum D; Step 6: Spread the cleaned porcine peritoneum D on a freeze-drying plate, make sure the porcine peritoneum is as flat as possible, and freeze-dry it at -20°C for 24 hours to obtain the basic periosteum; Step 7: (1) Add 3 g of carboxymethyl chitosan to 100 mL of pure water and heat and stir to dissolve to obtain a carboxymethyl chitosan solution; (2) Evenly apply the carboxymethyl chitosan solution on the surface of the base periosteum with a coating amount of 0.2 g / cm 2; Spread it flat on a freeze-drying plate, keep the pig peritoneum as flat as possible, freeze-dry it at -20℃ for 24h to obtain the antibacterial periosteum.

[0024] Embodiment 4: A method for preparing an oral antibacterial periosteum, comprising the following steps: Step 1: Raw material preparation: pig film, carboxymethyl chitosan; Step 2: Pretreatment of pig peritoneum: remove large areas of fat from the pig peritoneum, trim the edges, and rinse with running pure water until there is no obvious blood stain or other attachments on the surface of the pig peritoneum, thereby obtaining pig peritoneum A; Step 3: Degreasing treatment: Use 0.09M sodium hydroxide solution to degrease and clean the membrane A in a shaker (parameters: 30°C, 135rpm, 4 hours, 2 times); then clean it with pure water (parameters: 30°C, 135rpm, 20min, 5 times); then clean it with isopropanol (parameters: 40°C, 135rpm, 8h); obtain porcine peritoneum B; Step 4: Decellularization: Use 3M sodium chloride solution to decellularize and wash porcine peritoneum B in a shaker (parameters: room temperature, 135 rpm, 1 h); then wash with pure water (parameters: room temperature, 135 rpm, 0.5 h); obtain porcine peritoneum C; Step 5: Cleaning: The porcine peritoneum C is further cleaned with pure water (parameters: room temperature, 135 rpm, 0.5 h, 4 times), and then with ultrasound (parameters: room temperature, 20 min, 2 times) to obtain porcine peritoneum D; Step 6: Spread the cleaned porcine peritoneum D on a freeze-drying plate, make sure the porcine peritoneum is as flat as possible, and freeze-dry it at -20°C for 24 hours to obtain the basic periosteum; Step 7: (1) Add 4 g of carboxymethyl chitosan to 100 mL of pure water and heat and stir to dissolve to obtain a carboxymethyl chitosan solution; (2) Apply the carboxymethyl chitosan solution evenly on the surface of the base periosteum with a coating amount of 0.08 g / cm 2 ; Spread it flat on a freeze-drying plate, keep the pig peritoneum as flat as possible, freeze-dry it at -20℃ for 24h to obtain the antibacterial periosteum.

[0025] Embodiment 5: A method for preparing an oral antibacterial periosteum, comprising the following steps: Step 1: Raw material preparation: pig film, carboxymethyl chitosan; Step 2: Pretreatment of pig peritoneum: remove large areas of fat from the pig peritoneum, trim the edges, and rinse with running pure water until there is no obvious blood stain or other attachments on the surface of the pig peritoneum, thereby obtaining pig peritoneum A; Step 3: Degreasing treatment: Use 0.1M sodium hydroxide solution to degrease and clean the membrane A in a shaker (parameters: 30°C, 135rpm, 4 hours, 2 times); then clean it with pure water (parameters: 30°C, 135rpm, 20min, 5 times); then clean it with isopropanol (parameters: 40°C, 135rpm, 8h); obtain porcine peritoneum B; Step 4: Decellularization: Use 4M sodium chloride solution to decellularize and wash the porcine peritoneum B in a shaker (parameters: room temperature, 135 rpm, 1 h); then wash with pure water (parameters: room temperature, 135 rpm, 0.5 h); obtain porcine peritoneum C; Step 5: Cleaning: The porcine peritoneum C is further cleaned with pure water (parameters: room temperature, 135 rpm, 0.5 h, 4 times), and then with ultrasound (parameters: room temperature, 20 min, 2 times) to obtain porcine peritoneum D; Step 6: Spread the cleaned porcine peritoneum D on a freeze-drying plate, make sure the porcine peritoneum is as flat as possible, and freeze-dry it at -20°C for 24 hours to obtain the basic periosteum; Step 7: (1) Add 5 g of carboxymethyl chitosan to 100 mL of pure water and heat and stir to dissolve to obtain a carboxymethyl chitosan solution; (2) Apply the carboxymethyl chitosan solution evenly on the surface of the base periosteum with a coating amount of 0.1 g / cm 2 ; Spread it flat on a freeze-drying plate, keep the pig peritoneum as flat as possible, freeze-dry it at -20℃ for 24h to obtain the antibacterial periosteum.

[0026] Embodiment 6: A further solution based on Embodiment 2 specifically comprises the following steps: Step 1: Raw material preparation: pig film, carboxymethyl chitosan, 8kDa low molecular weight hyaluronic acid, 100kDa medium molecular weight hyaluronic acid; Step 2: Pretreatment of pig peritoneum: remove large areas of fat from the pig peritoneum, trim the edges, and rinse with running pure water until there is no obvious blood stain or other attachments on the surface of the pig peritoneum, thereby obtaining pig peritoneum A; Step 3: Degreasing treatment: Use 0.03M sodium hydroxide solution to degrease and clean the membrane A in a shaker (parameters: 30°C, 135rpm, 4 hours, 2 times); then clean it with pure water (parameters: 30°C, 135rpm, 20min, 5 times); then clean it with isopropanol (parameters: 40°C, 135rpm, 8h); obtain porcine peritoneum B; Step 4: Decellularization: Use 1M sodium chloride solution to decellularize and wash the porcine peritoneum B in a shaker (parameters: room temperature, 135 rpm, 1 h); then wash with pure water (parameters: room temperature, 135 rpm, 0.5 h); obtain porcine peritoneum C; Step 5: Cleaning: The porcine peritoneum C is further cleaned with pure water (parameters: room temperature, 135 rpm, 0.5 h, 4 times), and then with ultrasound (parameters: room temperature, 20 min, 2 times) to obtain porcine peritoneum D; Step 6: Spread the cleaned porcine peritoneum D on a freeze-drying plate, make sure the porcine peritoneum is as flat as possible, and freeze-dry it at -20°C for 24 hours to obtain the basic periosteum; Step 7: (1) adding 2 wt% carboxymethyl chitosan to 2 v / v% acetic acid solution and stirring evenly to obtain solution A; adding 0.3 wt% aldehyded hyaluronic acid and 0.7 wt% quaternized hyaluronic acid to pure water and stirring evenly to obtain solution B; mixing equal volumes of solution A and solution B evenly to obtain a coating solution; (2) adding 0.05 wt% hydrogen peroxide and 0.002 wt% laccase into pure water to obtain an interfacial layer solution; (3) Pre-spray the interface layer solution on the surface of the base periosteum at a spraying amount of 0.02 g / cm 2 ; Then the coating solution is evenly coated on its surface, and the coating amount is 0.3g / cm 2 ; Spread it on a freeze-drying plate and let it stand at room temperature for 1 hour; then pre-freeze it at -20℃ for 5 hours and freeze-dry it at -30℃ for 24 hours to obtain the antibacterial periosteum.

[0027] The preparation method of aldehyded hyaluronic acid is as follows: 1 part of low molecular weight hyaluronic acid is added to 100 parts of pure water and stirred evenly, 5 parts of 0.1 g / mL sodium periodate solution are added dropwise, stirred for 5 hours in a dark place, ethylene glycol is added for neutralization, dialyzed, and dried to obtain modified hyaluronic acid; under a nitrogen atmosphere, it is further added to pure water and stirred evenly, 1.5 parts of EDC and 0.9 parts of NHS are added and stirred evenly, 5 parts of 0.05 g / mL dopamine are added, stirred at room temperature for 4 hours, dialyzed, and dried to obtain aldehyded hyaluronic acid; The preparation method of quaternized hyaluronic acid is as follows: 10 parts of medium molecular weight hyaluronic acid are added to 200 parts of 50wt% formamide aqueous solution and stirred evenly, the temperature is raised to 60°C, 0.8 parts of sodium hydroxide solution and 8 parts of 2,3-epoxypropyltrimethylammonium chloride are added, the mixture is stirred for 4 hours, and the mixture is purified and dried to obtain quaternized hyaluronic acid.

[0028] Comparative Example 2: Based on a further solution of Example 6, the ratio of the two hyaluronic acids was adjusted, specifically comprising the following steps: Step 1: Raw material preparation: pig film, carboxymethyl chitosan, 8kDa low molecular weight hyaluronic acid, 100kDa medium molecular weight hyaluronic acid; Step 2: Pretreatment of pig peritoneum: remove large areas of fat from the pig peritoneum, trim the edges, and rinse with running pure water until there is no obvious blood stain or other attachments on the surface of the pig peritoneum, thereby obtaining pig peritoneum A; Step 3: Degreasing treatment: Use 0.03M sodium hydroxide solution to degrease and clean the membrane A in a shaker (parameters: 30°C, 135rpm, 4 hours, 2 times); then clean it with pure water (parameters: 30°C, 135rpm, 20min, 5 times); then clean it with isopropanol (parameters: 40°C, 135rpm, 8h); obtain porcine peritoneum B; Step 4: Decellularization: Use 1M sodium chloride solution to decellularize and wash the porcine peritoneum B in a shaker (parameters: room temperature, 135 rpm, 1 h); then wash with pure water (parameters: room temperature, 135 rpm, 0.5 h); obtain porcine peritoneum C; Step 5: Cleaning: The porcine peritoneum C is further cleaned with pure water (parameters: room temperature, 135 rpm, 0.5 h, 4 times), and then with ultrasound (parameters: room temperature, 20 min, 2 times) to obtain porcine peritoneum D; Step 6: Spread the cleaned porcine peritoneum D on a freeze-drying plate, make sure the porcine peritoneum is as flat as possible, and freeze-dry it at -20°C for 24 hours to obtain the basic periosteum; Step 7: (1) adding 2 wt% carboxymethyl chitosan to 2 v / v% acetic acid solution and stirring evenly to obtain solution A; adding 0.7 wt% aldehyded hyaluronic acid and 0.3 wt% quaternized hyaluronic acid to pure water and stirring evenly to obtain solution B; mixing equal volumes of solution A and solution B evenly to obtain a coating solution; (2) adding 0.05 wt% hydrogen peroxide and 0.002 wt% laccase into pure water to obtain an interfacial layer solution; (3) Pre-spray the interface layer solution on the surface of the base periosteum at a spraying amount of 0.02 g / cm 2 ; Then the coating solution is evenly coated on its surface, and the coating amount is 0.3g / cm 2 ; Spread it on a freeze-drying plate and let it stand at room temperature for 1 hour; then pre-freeze it at -20℃ for 5 hours and freeze-dry it at -30℃ for 24 hours to obtain the antibacterial periosteum.

[0029] Experiment 1: The periosteum and antibacterial periosteum prepared in Examples 1 to 5 and Comparative Example 1 were subjected to relevant scanning electron microscopy experiments, and the obtained images are as follows: Figure 1 to Figure 6 As shown; Conclusion: Figure 1 to Figure 6 The scanning electron microscope images show that compared with the comparative example 1, which has no special structure on the periosteum surface, the antibacterial periosteum of Examples 1 to 5 has a mesh-like surface due to carboxymethyl chitosan.

[0030] Experiment 2: The periosteum prepared in Examples 1 to 5 and Comparative Example 1 and the antibacterial periosteum were subjected to an antibacterial test. During the test, the periosteum was placed in a 12-well plate, and 15 μL of Staphylococcus aureus solution (1×10 6 CFU•mL -1 ) was added to the central area of ​​the periosteum. A 1cm×1cm polyethylene film was then covered on the bacterial solution. Afterwards, a small amount of PBS was added to the wells around the sample, and the 12-well plate was placed in a 37°C incubator to ensure the appropriate temperature and humidity required for normal bacterial growth. After 24 hours of incubation, 2mL of PBS buffer was added to each well, and the well plate was sonicated for 5 minutes to release bacteria adhered to the periosteum. Serial dilutions of PBS were then spread on agar plates and incubated for 24 hours before colony counting. The results are shown in the figure. Figure 7 The sterilization efficiency is shown in the figure. Among them, "*" represents P < 0.05, "**" represents P < 0.01, "***" represents P < 0.001, and "****" represents P < 0.0001.

[0031] Conclusion: As can be seen from the figure, the antibacterial periosteum coated with carboxymethyl chitosan has excellent bactericidal efficiency.

[0032] Experiment 3: The periosteum prepared in Examples 1 to 6 and Comparative Examples 1 to 2 and the antibacterial periosteum were subjected to a biocompatibility test. During the process, BMSCs (mouse bone marrow mesenchymal stem cells) were first cultured in a DMEM medium containing 10% fetal bovine serum. The periosteum was soaked in the above culture medium for 24 hours (6 cm 2 / mL), and the cells were plated at 1×10 4 The cells were inoculated into 96-well plates at a concentration of 100%, 75%, 50%, and 25% and cultured at 37°C in a humidified atmosphere with 5% CO2 for 24 hours. The periosteum extract was then diluted to 100%, 75%, 50%, and 25% and added to the 96-well plates for incubation with the cells for 24 hours. MTT reagent (40 μL) was then added and cultured at 37°C for 4 hours. The MTT reagent was then removed, and the purple crystals were dissolved with 100% dimethyl sulfoxide (DMSO, 100 μL), and the absorbance of the sample was measured using a multifunctional microplate reader (λ=570 nm). The results are shown in Figure 2. Figures 8 to 14 The significant differences were compared with the negative group, "*" represents P < 0.05, "**" represents P < 0.01, "***" represents P < 0.001, and "****" represents P < 0.0001.

[0033] Conclusion: As can be seen from the figure, the antibacterial periosteum coated with carboxymethyl chitosan has good biocompatibility and meets the needs of oral implant periosteum; at the same time, the biocompatibility can be further improved by further introducing two hyaluronic acid derivatives.

[0034] Experiment 4: Example 2, Example 6, and Comparative Example 2 were subjected to a toughness test at a rate of 10 mm / min. The obtained data are as follows:

[0035] Conclusion: From the data in the above table, it can be seen that the introduction of hyaluronic acid effectively enhances the mechanical properties.

[0036] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A method for preparing an antibacterial periosteum for oral use, characterized in that: The following steps are involved: Step 1: Prepare raw materials; Step 2: Pretreatment of pig peritoneum: remove large areas of fat from the pig peritoneum, trim the edges, and wash with pure water until there is no obvious attachment to obtain pig peritoneum A; Step 3: Degreasing treatment: placing the porcine peritoneum A in a sodium hydroxide solution for degreasing and washing, and then washing with pure water and isopropanol in sequence to obtain the porcine peritoneum B; Step 4: decellularization treatment: placing the porcine peritoneum B in a sodium chloride solution for decellularization and washing, and then washing with pure water to obtain porcine peritoneum C; Step 5: Cleaning: The porcine peritoneum C is further cleaned with pure water and then ultrasonically cleaned to obtain porcine peritoneum D; Step 6: Spread the porcine peritoneum D on a freeze-drying plate and freeze-dry it to obtain the basic periosteum; Step 7: (1) Add carboxymethyl chitosan to pure water and stir evenly to obtain a carboxymethyl chitosan solution with a concentration of 1-5 wt%; (2) Evenly coat the carboxymethyl chitosan solution on the surface of the base periosteum, spread it on a freeze-drying plate, and freeze-dry to obtain an antibacterial periosteum.

2. The method for preparing an oral antibacterial periosteum according to claim 1, characterized in that: In step 3, during the degreasing and cleaning process, the temperature is 28-32° C., the shaking speed is 130-140 rpm, the time is 3-5 hours, and the number of degreasing and cleaning times is 2-3 times; During the pure water washing process, the temperature is 28-32°C, the shaking speed is 130-140 rpm, the time is 15-25 minutes, and the number of pure water washings is 4-6 times; During the isopropanol cleaning process, the temperature is 38-42° C., the shaking speed is 130-140 rpm, the time is 7-9 hours, and the number of isopropanol cleanings is 2-3 times.

3. The method for preparing an oral antibacterial periosteum according to claim 1, characterized in that: In step 4, during the decellularization washing process, the temperature is room temperature, the shaking speed is 130-140 rpm, and the time is 1-1.5 hours; during the pure water washing process, the temperature is room temperature, the shaking speed is 130-140 rpm, and the time is 0.5-0.6 hours; In step 5, during the pure water cleaning process, the temperature is room temperature, the shaking speed is 130-140 rpm, the time is 0.5-0.6 hours, and the number of pure water cleanings is 3-5 times; during the ultrasonic cleaning process, the temperature is room temperature, the time is 15-20 minutes, and the number of ultrasonic cleanings is 2-3 times.

4. The method for preparing an oral antibacterial periosteum according to claim 1, characterized in that: In step 3, the concentration of the sodium hydroxide solution is 0.01M~0.1M.

5. The method for preparing an oral antibacterial periosteum according to claim 1, characterized in that: In step 4, the concentration of the sodium chloride solution is 0.5M~4M.

6. The method for preparing an oral antibacterial periosteum according to claim 1, characterized in that: In step 7, the coating amount of the carboxymethyl chitosan solution is 0.05-0.5 g / cm 2 .

7. The method for preparing an oral antibacterial periosteum according to claim 1, characterized in that: The steps in step 7 are: (1) adding 1.5-2.5 wt% of carboxymethyl chitosan to 1-2 v / v% of acetic acid solution and stirring evenly to obtain solution A; adding 0.2-0.4 wt% of aldehyded hyaluronic acid and 0.6-0.8 wt% of quaternary ammonium hyaluronic acid to pure water and stirring evenly to obtain solution B; mixing equal volumes of solution A and solution B evenly to obtain a coating solution; (2) adding 0.05-0.1 wt% hydrogen peroxide and 0.002-0.004 wt% laccase into pure water to obtain an interfacial layer solution; (3) Pre-spray the interface layer solution on the surface of the base periosteum at a spraying amount of 0.02-0.03 g / cm 2 ; Then evenly apply the coating solution on its surface, the coating amount is 0.25~0.35g / cm 2 ; Spread it flat on a freeze-drying plate and let it stand at room temperature for 1 to 2 hours; then pre-freeze it at -20℃~-25℃ for 4 to 6 hours, and freeze-dry it at -50℃~-60℃ for 24 to 48 hours to obtain the antibacterial periosteum.

8. The method for preparing an oral antibacterial periosteum according to claim 7, characterized in that: The steps in step 7 are: the preparation method of the aldehyde-modified hyaluronic acid is: adding low molecular weight hyaluronic acid to pure water and stirring evenly, adding sodium periodate solution dropwise, stirring for 4 to 6 hours in a dark environment, adding ethylene glycol for neutralization, dialyzing, and drying to obtain modified hyaluronic acid; under a nitrogen atmosphere, further adding it to pure water and stirring evenly, adding EDC and NHS and stirring evenly, adding dopamine, stirring at room temperature for 3 to 4 hours, dialyzing, and drying to obtain aldehyde-modified hyaluronic acid; The preparation method of the quaternary ammonium hyaluronic acid is as follows: adding a medium molecular weight hyaluronic acid to a 50-60wt% formamide aqueous solution and stirring evenly, heating to 60-65° C., adding a sodium hydroxide solution and 2,3-epoxypropyltrimethylammonium chloride, stirring for reaction for 3-4 hours, purifying, and drying to obtain the quaternary ammonium hyaluronic acid.

9. The method for preparing an oral antibacterial periosteum according to claim 8, characterized in that: The average molecular weight of the hyaluronic acid is 8-10 kDa; the average molecular weight of the medium molecular hyaluronic acid is 100-150 kDa.

10. The antibacterial periosteum prepared according to the method for preparing an oral antibacterial periosteum according to any one of claims 1 to 9.

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