Preparation method of oral keratinized gingival regeneration and repair stent and product thereof

Abstract

The invention relates to a preparation method of an oral keratinized gingival regeneration and repair stent and a product thereof, and the preparation method comprises the following steps: sequentially treating an acellular matrix with a first modifier, a fixing agent and a second modifier to obtain an enhanced acellular matrix; mixing the carrier solution with the antibacterial natural product solution, dropwise adding an ionic cross-linking agent, and centrifuging to obtain a precipitate, namely the drug-loaded nanoparticles; and mixing the enhanced acellular matrix, the drug-loaded nanoparticles and the covalent linking agent, washing with water, and freeze-drying to obtain the drug-loaded enhanced acellular matrix. The repair stent prepared by the invention has excellent biocompatibility, long-term stable degradation, outstanding antibacterial and anti-inflammatory effects and good mechanical properties, and can promote wound healing and revascularization.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and relates to a preparation method of an oral keratinized gingiva regeneration and repair scaffold and a product thereof. BACKGROUND

[0002] With the rapid development of oral implant technology, implant restoration has gradually become the preferred treatment for dentition defects and edentulism. Adequate and good quality of gingival tissue in the implant area is a key factor to ensure the success of implant restoration. It is generally believed in the clinic that, in order to maintain the long-term stability and good aesthetics of the implant, the width of the keratinized gingiva around the implant should be at least 2 mm, and the keratinized gingiva with a thickness of more than 2 mm can improve the marginal stability. However, in clinical practice, due to factors such as periodontal disease, systemic disease, poor oral hygiene habits, etc., the loss of gingival soft tissue attachment and frequent tissue trauma occur.

[0003] In the current clinical practice, the scheme of reposition flap surgery combined with autologous free gingival tissue or epithelial submucosa transplantation is mainly used to treat gingival soft tissue loss or deficiency. However, the autologous tissue donor site is limited, the implant area is prone to form scar-like texture, and there are postoperative complications such as pain, swelling and bleeding. In addition, the development of the second surgical area brings double pain to the patient. Therefore, it is particularly important to develop a guide keratinized gingiva regeneration and repair scaffold as a substitute for autologous tissue transplantation.

[0004] An ideal autologous tissue graft substitute should have the following characteristics: first, it should have good biocompatibility, promote wound healing and vascular regeneration in the early stage of implantation. Second, it should have long-term stable degradation characteristics to stimulate tissue regeneration for a long time, avoiding the problem of collapse and atrophy of the implanted area due to rapid absorption. Third, it should have good antibacterial and anti-inflammatory effects to resist the invasion of oral pathogenic bacteria and reduce the incidence of tissue inflammation. Finally, it should have good mechanical properties to reduce mechanical trauma and maintain tissue regeneration space for a long time. However, existing commercial products and technologies cannot simultaneously meet the above characteristics. CN 118892582B provides a preparation method of keratinized gingiva repair scaffold, disperses the silver / magnesium / zinc ion-doped bioactive glass microsphere powder in the purified collagen solution to obtain a loose surface stock solution, and freeze-dries the decellularized matrix together. After two cross-linking, a keratinized gingiva regeneration repair scaffold with a loose surface and a dense surface is obtained. However, the purified collagen has low mechanical strength and a rapid degradation rate, which is not conducive to long-term stable stimulation of tissue regeneration and maintenance of tissue regeneration space in the implanted area. In addition, although the added bioactive glass microsphere powder has antibacterial effect, it is only physically mixed with the main scaffold and not stably combined, which can easily separate after implantation, has the limitation of burst release, and leads to a short period of antibacterial ability. And the burst release leads to the release of a high concentration of silver / magnesium / zinc metal ions in the bioactive glass microspheres for a short period of time, which has a high risk of cytotoxicity and tissue damage, resulting in a series of complications.

[0005] Therefore, there is an urgent need to develop a new keratinized gingiva regeneration repair scaffold with good biocompatibility, stable degradation rate, promotion of wound healing and vascular regeneration, as well as good mechanical properties, antibacterial properties and anti-inflammatory effects. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide a preparation method of an oral keratinized gingiva regeneration repair scaffold and the product thereof.

[0007] To achieve the purpose of the present application, the following technical solutions are adopted:

[0008] In a first aspect, the present application provides a preparation method of an oral keratinized gingiva regeneration repair scaffold, which comprises:

[0009] (1) treating the decellularized matrix with a first modifier, a fixing agent and a second modifier in sequence to obtain an enhanced decellularized matrix;

[0010] After mixing the carrier solution and the antibacterial natural product solution, the ion cross-linking agent is added dropwise, and the obtained precipitate after centrifugation is the drug-loaded nanoparticles;

[0011] (2) mixing the enhanced decellularized matrix, drug-loaded nanoparticles and covalent coupling agent, washing with water, and freeze-drying to obtain the product.

[0012] The main structure of the keratinized gingiva regeneration and repair scaffold is a decellularized matrix, which has a rich interconnected pore structure, is conducive to nutrient transport and cell ingrowth, and effectively completes tissue fusion. The obtained keratinized gingiva regeneration and repair scaffold is mechanically enhanced, can effectively reduce mechanical trauma to the tissue, has outstanding anti-enzymatic properties, can stably maintain the space of the implanted area for a long time, and can stimulate keratinized gingiva tissue regeneration for a long time. The obtained scaffold is modified by adding an amino acid modifier, which promotes early cell adhesion and promotes early wound healing and vascular regeneration, and improves biocompatibility. The obtained scaffold is covalently connected to antibacterial nanoparticles, which are stable in combination with the slow release of the nanoparticles, and together with the slow release of the nanoparticles, the scaffold has long-term stable antibacterial ability. In addition, the antibacterial natural product therein also has multiple effects of anti-inflammatory, antioxidant, free radical scavenging, and promoting wound healing, ensuring that the obtained scaffold has excellent keratinized gingiva regeneration guiding ability.

[0013] Preferably, the first modifier and the second modifier independently comprise any one or a combination of at least two of threonine, serine, lysine, arginine, glutamic acid, aspartic acid, and cysteine.

[0014] Preferably, the fixing agent comprises any one or a combination of at least two of glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, dicyclohexyl carbodiimide, genipin, and 1.4 butanediol diglycidyl ether.

[0015] Preferably, the mass ratio of the decellularized matrix to the first modifier is (0.5-10):1, and the specific point values in (0.5-10) can be selected from 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0016] Preferably, the mass ratio of the decellularized matrix to the fixing agent is (0.1-10):1, and the specific point values in (0.1-10) can be selected from 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0017] Preferably, the mass ratio of the decellularized matrix to the second modifier is 1:(0.2-5), and each specific point value in the range of (0.2-5) can be selected as 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc., and other specific point values in the above numerical range can be selected.

[0018] Preferably, the treatment is carried out under the condition of a mixed medium, and the mixed medium includes PBS buffer, deionized water, MES buffer or sodium chloride solution.

[0019] Preferably, the solid-liquid ratio of the decellularized matrix to the mixed medium is 1 g:(10-500) mL, and each specific point value in the range of (10-500) can be selected as 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, etc., and other specific point values in the above numerical range can be selected.

[0020] Preferably, the temperature of the treatment is independently 4-45℃, and the time of a single treatment is 0.5-48 h.

[0021] The temperature can be selected as 4℃, 8℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, etc., and the time can be selected as 0.5 h, 1 h, 2 h, 5 h, 8 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h, 48 h, etc., and other specific point values in the above numerical range can be selected.

[0022] Preferably, the number of treatments is independently 1-6 times, such as 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, etc.

[0023] Preferably, the carrier in the carrier solution includes chitosan, and the solvent includes an acetic acid aqueous solution.

[0024] Chitosan has abundant primary amine groups, which can form nanoparticles by ionic cross-linking with negatively charged sodium tripolyphosphate through electrostatic interaction, thereby achieving the encapsulation of hydrophobic substances.

[0025] Preferably, the mass percentage of acetic acid in the acetic acid aqueous solution is 0.5-3%, such as 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8%, 3%, etc., and other specific point values in the above numerical range can be selected.

[0026] Preferably, the concentration of the carrier in the carrier solution is 0.2-5 mg / mL, for example 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, and the like, and other specific point values within the above numerical range can be selected, which will not be repeated here.

[0027] Preferably, the natural antibacterial product includes any one or a combination of at least two of curcumin, quercetin, and resveratrol.

[0028] Curcumin, quercetin, and resveratrol are all natural antibacterial substances, and extensive research has shown that they have antibacterial, anti-inflammatory, antioxidant, and wound healing effects, and are biologically safe. All of them are hydrophobic substances and can be coated by chitosan nanocarriers.

[0029] Preferably, the solvent of the natural antibacterial product solution includes ethanol, glacial acetic acid, or ethyl acetate.

[0030] Preferably, the volume ratio of the natural antibacterial product solution to the carrier solution is (0.1-5):1, and the specific point values in (0.1-5) can be selected as 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, and the like, and other specific point values within the above numerical range can be selected, which will not be repeated here.

[0031] Preferably, the concentration of the natural antibacterial product in the natural antibacterial product solution is 0.2-5 mg / mL, for example 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, and the like, and other specific point values within the above numerical range can be selected, which will not be repeated here.

[0032] Preferably, the ionic crosslinking agent includes a sodium tripolyphosphate aqueous solution.

[0033] Preferably, the concentration of sodium tripolyphosphate in the aqueous sodium tripolyphosphate solution is 0.2-100 mg / mL, such as 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, and the like, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0034] Preferably, the reaction endpoint of the ion crosslinking agent dropping is that the opalescence phenomenon appears and does not disappear.

[0035] Preferably, the speed of the centrifugation is 8000-12000 rpm, the time is 10-20 min, and the temperature is 4-26 ℃.

[0036] The speed can be selected as 8000 rpm, 8500 rpm, 9000 rpm, 9500 rpm, 10000 rpm, 10500 rpm, 11000 rpm, 11500 rpm, 12000 rpm, and the like, the time can be selected as 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, and the like, and the temperature can be selected as 4 ℃, 8 ℃, 12 ℃, 16 ℃, 20 ℃, 24 ℃, 26 ℃, and the like, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0037] Preferably, the obtained precipitate after centrifugation in step (1) is further washed with a cleaning agent, and the number of washing times is 1-3 times, such as 1 time, 2 times, 3 times.

[0038] Preferably, the cleaning agent includes a methanol aqueous solution with a mass percentage of 5-20%, such as 5%, 8%, 10%, 12%, 15%, 18%, 20%, and the like, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0039] Preferably, the covalent linker includes any one or a combination of at least two of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, dicyclohexyl carbodiimide.

[0040] The selected covalent linker can combine the carboxyl of the decellularized matrix with the amino of the chitosan nanoparticles through a covalent bond, thereby realizing stable combination of the nanoparticles and the decellularized matrix.

[0041] Preferably, the ratio of the enhanced decellularized matrix to the covalent linker is 1 g:(0.001-0.1) mol.

[0042] The specific point values ​​in (0.001-0.1) can be selected from 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0043] Preferably, the mass ratio of drug-loaded nanoparticles to enhanced decellularized matrix is ​​(0.001-0.05):1. Specific values ​​within (0.001-0.05) can be selected from 0.001, 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, etc. Other specific values ​​within the above range can also be selected, and will not be elaborated here.

[0044] Preferably, the mixing in step (2) is carried out under solution conditions, and the solvent includes deionized water, PBS buffer, MES buffer, and physiological saline.

[0045] Preferably, the solid-liquid ratio of the mixture in step (2) is 1:(10-500). The specific point values ​​in (10-500) can be selected from 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0046] Preferably, the mixing in step (2) is performed 1-4 times, the mixing temperature is 4-45 ℃, and the mixing time for each mixing is 0.5-48 h.

[0047] The number of times can be selected as 1, 2, 3, or 4 times; the temperature can be selected as 4℃, 8℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, or 45℃; and the time can be selected as 0.5 h, 1 h, 2 h, 5 h, 8 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h, or 48 h. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0048] Preferably, the method for preparing the decellularized matrix includes:

[0049] (1) Mix the skin tissue with an alkaline solution to remove hair, epidermis and subcutaneous tissue to obtain the dermis;

[0050] (2) Mix the dermis with an alkaline solution and wash to obtain the product;

[0051] The concentration gradient of the alkaline solution in step (2) is 1-5.

[0052] Preferably, the mass percentage of the base in the base solution in step (1) is 1-10%, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, and the like, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0053] Preferably, the ratio of the skin tissue to the base solution in step (1) is 1 g:(4-20) mL, and the specific point values in (4-20) can be selected as 4, 8, 12, 16, 20, and the like, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0054] Preferably, the temperature of the mixture in step (1) is 4-42 ℃, and the single treatment time is 0.5-48 h.

[0055] The temperature can be selected as 4 ℃, 8 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 42 ℃, and the like, and the time can be selected as 0.5 h, 1 h, 2 h, 5 h, 8 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h, 48 h, and the like, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0056] Preferably, the skin tissue includes human skin, cow skin, sheep skin, pig skin, donkey skin, horse skin, rabbit skin, or mouse skin.

[0057] Preferably, the concentration of each base solution is not higher than that of the previous base solution.

[0058] Preferably, the mass percentage of the base in the base solution in step (2) is 0.5-4%, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and the like, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0059] Preferably, the ratio of the dermis layer to the base solution in step (2) is 1 g:(4-20) mL, and the specific point values in (4-20) can be selected as 4, 8, 12, 16, 20, and the like, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0060] Preferably, the temperature of the treatment in step (12) is 4-45 ℃, and the single treatment time is 1-24 h.

[0061] The temperature can be selected from 4℃, 8℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, etc., and the time can be selected from 0.5 h, 1 h, 2 h, 5 h, 8 h, 10 h, 15 h, 20 h, 24 h, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0062] Preferably, the base includes sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, ammonia water, and further preferably sodium hydroxide and potassium hydroxide.

[0063] Preferably, the cleaning specifically includes cleaning the dermal layer to neutral, and the reagent used in the cleaning includes one or more of PBS buffer, deionized water, sodium chloride solution or calcium chloride solution.

[0064] In a second aspect, the present application provides an oral keratinized gingiva regeneration repair scaffold prepared by the preparation method of the oral keratinized gingiva regeneration repair scaffold according to the first aspect.

[0065] The keratinized gingiva regeneration repair scaffold has a thickness of 0.5-6 mm, a tensile strength of 6-12 Mpa, a 24 h enzyme treatment degradation residual rate of greater than 80%, and an antibacterial rate of greater than 90%. The keratinized gingiva regeneration repair scaffold loaded with the antibacterial component is a nano-particle structure, and the two are covalently bonded.

[0066] Compared with the prior art, the present application has the following beneficial effects:

[0067] The main structure of the keratinized gingiva regeneration repair scaffold of the present application is a decellularized matrix, which has a rich interconnected pore structure, is beneficial to nutrient transport and cell ingrowth, and effectively completes tissue fusion. The obtained keratinized gingiva regeneration repair scaffold is mechanically enhanced, can effectively reduce mechanical trauma to the tissue, has outstanding enzyme resistance, can stably maintain the space of the implanted area for a long time, and can stably stimulate the regeneration of keratinized gingiva tissue for a long time. The obtained scaffold is modified by adding an amino acid modifier, which promotes early cell adhesion and promotes early wound healing and vascular regeneration, and improves biocompatibility. The obtained scaffold is covalently connected with antibacterial nanoparticles, and the two are stably combined. The slow release of the nanoparticles together with the long-term stable antibacterial ability of the scaffold, in addition, the antibacterial natural product therein also has multiple effects of anti-inflammatory, antioxidant, free radical scavenging, and promoting wound healing, ensuring that the obtained scaffold has excellent guiding ability of keratinized gingiva regeneration. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 The oral keratinized gingiva regeneration repair scaffold prepared in Example 1 is shown in the figure.

[0069] Figure 2 The longitudinal section H&E staining diagram of the oral keratinized gingiva regeneration repair scaffold prepared in Example 1 is shown in the figure.

[0070] Figure 3 Longitudinal section micro-morphology electron microscope graph of the oral keratinized gingiva regeneration repair scaffold prepared in Example 1. DETAILED DESCRIPTION

[0071] The technical solutions of the present application are further illustrated by the specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

[0072] The efficacy ingredients in the products involved in the following examples and comparative examples are derived from the following sources (only the efficacy ingredients are embodied, and the necessary auxiliary ingredients contained in other commercially available raw materials are not described again):

[0073] Chitosan is a product with the trade name of chitosan purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd.;

[0074] Sodium tripolyphosphate is a product with the trade name of sodium tripolyphosphate purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd.;

[0075] Sodium hyaluronate is a product with the trade name of sodium hyaluronate purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.;

[0076] Polyvinyl alcohol is a product with the trade name of polyvinyl alcohol 1788 type purchased from Shanghai Maikelin Biochemical Technology Co., Ltd.

[0077] Example 1

[0078] The present embodiment provides a preparation method of a keratinized gingiva regeneration repair scaffold, which comprises:

[0079] (1) Obtain bovine skin from a slaughterhouse, wash off blood and impurities with water, cut into appropriate size, and then place in a polypropylene bottle ①. Then add 3% sodium hydroxide solution according to a solid-liquid ratio of 1:6, and tighten the bottle cap. Place the polypropylene bottle ① in a constant temperature shaker for 8 h (25 ℃, 150 rpm). Then take out the polypropylene bottle ①, replace it with new 2% sodium hydroxide solution according to the same proportion, and then place it in a 37 ℃ constant temperature water bath for 2 h. Then take out the bovine skin from the polypropylene bottle ①, and use a handheld scraper to easily remove the attached layers such as hair, epidermis, subcutaneous fat, etc., to obtain an intact dermis layer.

[0080] (2) The dermis layer is transferred into a clean polypropylene bottle ②, 1% sodium hydroxide solution is added according to a solid-liquid ratio of 1:6, the bottle cap is tightened, and the bottle is continuously placed in a constant temperature shaking incubator for oscillation for 16 h (25°C, 50 rpm). Then, the polypropylene bottle ② is taken out, and new 0.5% sodium hydroxide solution is added according to the same proportion, and the bottle is placed in a constant temperature shaking incubator for oscillation for 12 h (25°C, 50 rpm). Then, the bovine dermis is taken out from the polypropylene bottle ②, and washed with 1×PBS solution until neutral, to obtain a decellularized bovine dermal matrix.

[0081] (3) The decellularized bovine dermal matrix is transferred into a clean polypropylene bottle ③, and lysine is added according to a mass ratio of 1:1, and MES buffer is added according to a solid-liquid ratio of 1:50, and oscillation is performed in a constant temperature shaking incubator for 2 h (25°C, 100 rpm). After oscillation, the solution is discarded, lysine and MES buffer are added according to the same proportion, and oscillation is performed in a constant temperature shaking incubator for 4 h (25°C, 100 rpm).

[0082] (4) After oscillation, the solution is not discarded, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, N-hydroxysuccinimide are added in the polypropylene bottle ③, and the mass ratio of the two to the decellularized bovine dermal matrix is 2:1 and 1:1 respectively, and oscillation is performed in a constant temperature shaking incubator for 24 h (25°C, 100 rpm).

[0083] (5) After oscillation, the solution is discarded, threonine is added in the polypropylene bottle ③ according to a mass ratio of 1:1, and deionized water is added according to a solid-liquid ratio of 1:100, and oscillation is performed in a constant temperature shaking incubator for 2 h (25°C, 100 rpm). After oscillation, the solution is discarded, deionized water is added according to the same proportion, and oscillation is performed in a constant temperature shaking incubator for 2 h (25°C, 100 rpm), and the operation is repeated twice, to obtain a mechanically enhanced decellularized bovine dermal matrix.

[0084] (6) Chitosan is dissolved in 1% acetic acid aqueous solution to prepare a chitosan solution with a concentration of 1 mg / mL. Under light-proof conditions, curcumin is dissolved in anhydrous ethanol to prepare a curcumin solution with a concentration of 1 mg / mL. Sodium tripolyphosphate is dissolved in deionized water to prepare a sodium tripolyphosphate solution with a concentration of 10 mg / mL. Under constant stirring, the curcumin solution is slowly added to the chitosan solution, and the volume ratio of the chitosan solution to the curcumin solution is 1:1. Then, the sodium tripolyphosphate aqueous solution is slowly added thereto, until a milky light phenomenon appears and does not disappear after stirring. Centrifugation (4°C, 12000 rpm, 10 min) is performed, the supernatant is discarded, and the product is washed with 10% methanol aqueous solution three times, and the freeze-dried product is collected.

[0085] (7) Put the mechanically reinforced acellular bovine dermal matrix into a clean polypropylene bottle ④, and add the freeze-dried product according to a mass ratio of 0.01:1 (freeze-dried product: mechanically reinforced acellular bovine dermal matrix), 0.01 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide per milligram of mechanically reinforced acellular matrix. Add MES buffer according to a solid-liquid ratio of 1:50, and culture in a shaking incubator for 4 h (25 ℃, 100 rpm).

[0086] (8) After oscillation, wash with deionized water, freeze in a -60 ℃ ultra-low temperature refrigerator for 2 h, and freeze dry for 24 h to obtain the oral keratinized gingiva regeneration repair scaffold.

[0087] Example 2

[0088] The present embodiment provides a preparation method of a keratinized gingiva regeneration repair scaffold, which comprises:

[0089] (1) Obtain bovine skin from a slaughterhouse, wash off blood and impurities with water, cut into an appropriate size, and then place in a polypropylene bottle ①. Add 5% sodium hydroxide solution according to a solid-liquid ratio of 1:4, and tighten the bottle cap. Place the polypropylene bottle ① in a constant temperature shaker for 4 h (20 ℃, 150 rpm). Then replace the 5% sodium hydroxide solution with a new 2% sodium hydroxide solution according to the same proportion, and place it in a 20 ℃ constant temperature water bath for 2 h. Then take out the bovine skin from the polypropylene bottle ①, and use a handheld scraper to easily remove the hair, epidermis, subcutaneous fat and other accessory layers to obtain an intact dermis layer.

[0090] (2) Transfer the above-mentioned dermis layer into a clean polypropylene bottle ②, add 1% sodium hydroxide solution according to a solid-liquid ratio of 1:8, tighten the bottle cap, and continue to oscillate in a constant temperature oscillation incubator for 8 h (20 ℃, 50 rpm). Then replace the 1% sodium hydroxide solution with a new 0.5% sodium hydroxide solution according to the same proportion, and oscillate in a constant temperature shaker for 12 h (20 ℃, 50 rpm). Then take out the bovine dermis from the polypropylene bottle ②, and wash it thoroughly with 1% calcium chloride aqueous solution and deionized water until it is neutral to obtain an acellular bovine dermal matrix.

[0091] (3) Transfer the acellular bovine dermal matrix into a clean polypropylene bottle ③, add arginine according to a mass ratio of 1:2 (acellular bovine dermal matrix: arginine), and add MES buffer according to a solid-liquid ratio of 1:50, and oscillate in a constant temperature oscillation incubator for 1 h (25 ℃, 100 rpm).

[0092] (4) After oscillation, the solution was discarded, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, N-hydroxysuccinimide were added into polypropylene bottle ③, and the mass ratio of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, N-hydroxysuccinimide to acellular bovine dermal matrix was 2:1, 1:1 respectively, and oscillation was carried out in a constant temperature oscillation incubator for 8 h (37 ℃, 100 rpm).

[0093] (5) After oscillation, the solution was discarded, 1:1 mass ratio of serine was added into polypropylene bottle ③, deionized water was added at a solid-liquid ratio of 1:100, and oscillation was carried out in a constant temperature oscillation incubator for 24 h (25 ℃, 100 rpm). After oscillation, the solution was discarded, deionized water was added at the same solid-liquid ratio, and oscillation was carried out in a constant temperature oscillation incubator for 12 h (25 ℃, 100 rpm), and the operation was repeated twice, to obtain the mechanical enhanced acellular bovine dermal matrix.

[0094] (6) Chitosan was dissolved in 1% acetic acid aqueous solution to prepare a chitosan solution with a concentration of 0.8 mg / mL. Under light-proof conditions, quercetin was dissolved in anhydrous ethanol to prepare a quercetin solution with a concentration of 0.8 mg / mL. Sodium tripolyphosphate was dissolved in deionized water to prepare a sodium tripolyphosphate solution with a concentration of 20 mg / mL. Under constant stirring conditions, the quercetin solution was slowly added to the chitosan solution, and the volume ratio of the chitosan solution to the quercetin solution was 1:2, and then the sodium tripolyphosphate aqueous solution was slowly added, until a milky light phenomenon appeared and did not disappear after stirring. Centrifugation (4 ℃, 12000 rpm, 10 min), discard the supernatant, wash with 10% methanol aqueous solution three times, and collect the freeze-dried product.

[0095] (7) The mechanical enhanced acellular bovine dermal matrix was placed in a clean polypropylene bottle ④, and the freeze-dried product was added at a mass ratio of 0.05:1 (freeze-dried product:mechanical enhanced acellular bovine dermal matrix). Each milligram of mechanical enhanced acellular matrix was added with 0.02 mmol of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, N-hydroxysuccinimide. MES buffer was added at a solid-liquid ratio of 1:100, and incubation was carried out in a shaking incubator for 12 h (37 ℃, 100 rpm).

[0096] (8) After oscillation, deionized water was used for washing, and the product was frozen in a -60 ℃ ultra-low temperature refrigerator for 2 h, and freeze-dried for 24 h, to obtain the oral keratinized gingiva regeneration repair scaffold.

[0097] Example 3

[0098] The present embodiment provides a preparation method of a keratinized gingiva regeneration repair scaffold, and the preparation method is as follows:

[0099] (1) Obtain bovine skin from slaughterhouse, rinse with water to remove blood and impurities, cut into appropriate size and place in polypropylene bottle 1, then add 3% sodium hydroxide solution according to the solid-liquid ratio of 1:8, and tighten the bottle cap. Place the polypropylene bottle 1 in a constant temperature shaker for 6 h (25℃, 150 rpm). Then take out the polypropylene bottle 1, replace it with new 2% sodium hydroxide solution according to the same proportion, and place it in a 25℃ constant temperature water bath for 4 h. Then take out the bovine skin from the polypropylene bottle 1, and use a handheld scraper to easily remove the hair, epidermis, subcutaneous fat and other accessory layers to obtain the intact dermis layer.

[0100] (2) Transfer the above dermis layer into a clean polypropylene bottle 2, add 1% sodium hydroxide solution according to the solid-liquid ratio of 1:5, tighten the bottle cap, and continue to place it in a constant temperature shaking incubator for 4 h (25℃, 50 rpm). Then take out the polypropylene bottle 2, replace it with new 0.5% sodium hydroxide solution according to the same proportion, and place it in a constant temperature shaker for 24 h (20℃, 50 rpm). Then take out the bovine dermis from the polypropylene bottle 2, and wash it with 1 M and 0.1 M sodium chloride solution alternately until it is neutral, to obtain the decellularized bovine dermis matrix.

[0101] (3) Transfer the decellularized bovine dermis matrix into a clean polypropylene bottle 3, add glutamic acid according to the mass ratio of 1:0.5 (decellularized bovine dermis matrix: glutamic acid), and add PBS buffer according to the solid-liquid ratio of 1:50, and shake in a constant temperature shaking incubator for 4 h (25℃, 100 rpm).

[0102] (4) After shaking, do not discard the solution, add glutaraldehyde in polypropylene bottle 3, and the mass ratio of glutaraldehyde to decellularized bovine dermis matrix is 0.2:1, and shake in a constant temperature shaking incubator for 4 h (37℃, 100 rpm).

[0103] (5) After shaking, discard the solution, add lysine in polypropylene bottle 3 according to the mass ratio of 1:1, and add deionized water according to the solid-liquid ratio of 1:100, and shake in a constant temperature shaking incubator for 24 h (25℃, 100 rpm). After shaking, discard the solution, add deionized water according to the same solid-liquid ratio, and shake in a constant temperature shaking incubator for 6 h (25℃, 100 rpm), and repeat the operation three times to obtain the mechanically enhanced decellularized bovine dermis matrix.

[0104] (6) 1% acetic acid aqueous solution was used to dissolve chitosan to prepare a chitosan solution with a concentration of 1 mg / mL. Resveratrol was dissolved in anhydrous ethanol under light-proof conditions to prepare a resveratrol solution with a concentration of 1.5 mg / mL. Sodium tripolyphosphate was dissolved in deionized water to prepare a sodium tripolyphosphate solution with a concentration of 10 mg / mL. Under constant stirring, the resveratrol solution was slowly added to the chitosan solution, and the volume ratio of the chitosan solution to the resveratrol solution was 1:1, and then the sodium tripolyphosphate aqueous solution was slowly added thereto until a milky light appeared and did not disappear after stirring. Centrifugation (4 ℃, 12000 rpm, 10 min) was performed, the supernatant was discarded, 10% methanol aqueous solution was used for washing three times, and the freeze-dried product was collected.

[0105] (7) The mechanically enhanced acellular bovine dermal matrix was placed in a clean polypropylene bottle ④, and the freeze-dried product was added according to a mass ratio of 0.01:1 (freeze-dried product: mechanically enhanced acellular bovine dermal matrix), and 0.01 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide was added per milligram of mechanically enhanced acellular matrix. MES buffer was added according to a solid-liquid ratio of 1:50, and incubation was performed in a shaking incubator for 8 h (37 ℃, 100 rpm).

[0106] (8) After shaking, deionized water was used for washing, and the product was frozen in a -60 ℃ ultra-low temperature refrigerator for 2 h, and freeze-dried for 24 h to obtain the oral keratinized gingiva regeneration and repair scaffold.

[0107] Example 4

[0108] The present embodiment provides a preparation method of a keratinized gingiva regeneration and repair scaffold, which is different from example 1 only in that step (3) is "the acellular bovine dermal matrix is transferred into a clean polypropylene bottle ③, and sodium hyaluronate is added, and the mass ratio of the acellular bovine dermal matrix to the sodium hyaluronate is 1:0.2, and MES buffer is added according to a solid-liquid ratio of 1:50, and shaking is performed in a constant-temperature shaking incubator for 2 h (25 ℃, 100 rpm)", and step (5) is "after shaking, the solution is discarded, sodium hyaluronate is added in the polypropylene bottle ③, the mass ratio of the acellular bovine dermal matrix to the sodium hyaluronate is 1:0.2, deionized water is added according to a solid-liquid ratio of 1:100, and shaking is performed in a constant-temperature shaking incubator for 2 h (25 ℃, 100 rpm). After shaking, the solution is discarded, deionized water is added according to the same proportion, and shaking is performed in a constant-temperature shaking incubator for 2 h (25 ℃, 100 rpm), and the operation is repeated twice to obtain the mechanically enhanced acellular bovine dermal matrix". The other operations remain unchanged.

[0109] Example 5

[0110] The embodiment provides a preparation method of a keratinized gingiva regeneration repair support, which is different from example 1 only in that step (3) is "put the acellular bovine dermal matrix into a clean polypropylene bottle ③, add polyvinyl alcohol, the mass ratio of the acellular bovine dermal matrix to the polyvinyl alcohol is 1:0.2, add MES buffer solution at a solid-liquid ratio of 1:100, and oscillate in a constant-temperature oscillation incubator for 2 h (25 ℃, 100 rpm). After oscillation, the solution is discarded, the same proportion of polyvinyl alcohol and MES buffer solution is added, and oscillation is carried out in a constant-temperature oscillation incubator for 4 h (25 ℃, 100 rpm). After oscillation, the solution is discarded, deionized water is added in the polypropylene bottle ③, the mass ratio of the acellular bovine dermal matrix to the polyvinyl alcohol is 1:0.2, deionized water is added at a solid-liquid ratio of 1:100, and oscillation is carried out in a constant-temperature oscillation incubator for 2 h (25 ℃, 100 rpm). After oscillation, the solution is discarded, deionized water is added at the same proportion, oscillation is carried out in a constant-temperature oscillation incubator for 2 h (25 ℃, 100 rpm), and the operation is repeated twice, to obtain the mechanically enhanced acellular bovine dermal matrix". Other operations remain unchanged.

[0111] Example 6

[0112] The embodiment provides a preparation method of a keratinized gingiva regeneration repair support, which is different from example 1 only in that step (7) is "put the mechanically enhanced acellular bovine dermal matrix into a clean polypropylene bottle ④, add the freeze-dried product at a mass ratio of 0.01:1 (freeze-dried product:mechanically enhanced acellular bovine dermal matrix), add 0.01 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide per milligram of mechanically enhanced acellular dermal matrix, add MES buffer solution at a solid-liquid ratio of 1:50, and culture in an oscillation incubator for 4 h (25 ℃, 100 rpm)", and other operations remain unchanged.

[0113] Example 7

[0114] The embodiment provides a preparation method of a keratinized gingiva regeneration repair support, which is different from example 1 only in that step (7) is "put the mechanically enhanced acellular bovine dermal matrix into a clean polypropylene bottle ④, add the freeze-dried product at a mass ratio of 0.01:1 (freeze-dried product:mechanically enhanced acellular bovine dermal matrix), add 0.01 mmol of glutaraldehyde per milligram of mechanically enhanced acellular dermal matrix, add PBS buffer solution at a feeding ratio of 1:50, and culture in an oscillation incubator for 4 h (25 ℃, 100 rpm)", and other operations remain unchanged.

[0115] The glutaraldehyde is selected as the covalent linker in the embodiment, which can act on the amino groups of the chitosan and the amino groups of the decellularized matrix respectively, and then covalently connect the drug-loaded nanoparticles and the enhanced decellularized matrix.

[0116] Comparative Example 1

[0117] The comparative example provides a preparation method of the keratinized gingiva regeneration repair scaffold, which is different from the embodiment 1 only in that the step (5) is "after the oscillation is completed, the solution is discarded, deionized water is added in the polypropylene bottle ③ at a solid-liquid ratio of 1:100, and oscillation is performed in a constant-temperature oscillation incubator for 2 h (25 ℃, 100 rpm). After the oscillation is completed, the solution is discarded, deionized water is added at the same ratio, and oscillation is performed in a constant-temperature oscillation incubator for 2 h (25 ℃, 100 rpm), and the operation is repeated twice, to obtain the mechanically enhanced decellularized bovine dermal matrix", and other operations remain unchanged. That is, the modifier is used for modification treatment only once.

[0118] Comparative Example 2

[0119] The comparative example provides a preparation method of the keratinized gingiva regeneration repair scaffold, which is different from the embodiment 1 only in that the step (7) is "the mechanically enhanced decellularized bovine dermal matrix is placed in a clean polypropylene bottle ④, the freeze-dried product is added at a mass ratio of 0.01:1 (freeze-dried product:mechanically enhanced decellularized bovine dermal matrix), deionized water is added at a solid-liquid ratio of 1:50, and incubation is performed in an oscillation incubator for 12 h (25 ℃, 100 rpm)", and other operations remain unchanged.

[0120] The chitosan carrier has a positively charged shell, which can be electrostatically combined with the negatively charged domains of the enhanced decellularized matrix. In addition, the chitosan carrier has abundant hydroxyl and amino groups, which can act as hydrogen bond donors, and the enhanced decellularized matrix has abundant hydrogen bond acceptors such as carbonyl groups, so the two can also be combined by hydrogen bonds. The comparative example combines the enhanced decellularized matrix and the drug-loaded nanoparticles through electrostatic interaction and hydrogen bond interaction.

[0121] Test Example 1

[0122] H&E Staining and Microscopic Morphology Observation

[0123] The oral keratinized gingiva regeneration repair scaffold prepared in the embodiment 1 is shown in Figure 1 , and the scaffold is white to light yellow. After H&E staining, clear collagen fiber structure is observed under an electron microscope Figure 2 , and the collagen fibers are stained red by eosin), the collagen fiber bundles are arranged densely, and no obvious disorder and damage of the collagen fiber bundle structure are observed.

[0124] The longitudinal section microscopic structure electron microscope graph of the oral keratinized gingiva regeneration repair scaffold prepared in the embodiment 1 is shown in Figure 3The longitudinal section structure of the stent presents a "loose-dense-loose" feature. The outer side of the loose structure is conducive to early tissue fusion and vascularization, and the inner side of the dense structure ensures that the stent can exist stably in the implanted area for a long time, and long-term stable stimulation of keratinized gingival tissue regeneration in the implanted area.

[0125] Test Example 2

[0126] Tensile strength test

[0127] The tensile strength of the oral keratinized gingiva regeneration repair stent is tested according to the standard GB / T 528-2009 "Determination of tensile stress-strain properties of vulcanized or thermoplastic rubber".

[0128] Table 1

[0129]

[0130] The oral keratinized gingiva regeneration repair stents prepared in Examples 1-3 all have high mechanical strength. The use of other modifiers, without secondary modification, the use of other covalent linking agents, and the use of other connection methods will all affect the tensile strength of the stent.

[0131] Test Example 3

[0132] Degradation performance test

[0133] The oral keratinized gingiva regeneration repair stents prepared in Examples 1-7 and Comparative Examples 1-2 are all cut into rectangular samples with a size of 1 cm x 2 cm, and the mass is precisely detected. Incubate with 80 U / mL of collagenase solution (0.01M Tris HCl system) for 24 h (37℃, 50 rpm), after incubation, wash the samples thoroughly with deionized water, freeze-dry, and precisely detect the mass. Calculate the percentage of the mass after enzyme treatment to the mass before enzyme treatment, which is the degradation residual rate.

[0134] Table 2

[0135]

[0136] After testing, the oral keratinized gingiva regeneration repair stents prepared in Examples 1-3 all have high enzymatic resistance. The use of other modifiers, without secondary modification, the use of other covalent linking agents, and the use of other connection methods will all affect the enzymatic resistance of the stent.

[0137] Test Example 4

[0138] Cytotoxicity test

[0139] The cytotoxicity detection is performed according to the standards GB / T 16886.12-2023 “Medical devices-Biological evaluation Part 12: Sample preparation and reference materials”, GB / T 16886.5-2017 “Medical devices-Biological evaluation Part 5: In vitro cytotoxicity tests”.

[0140] Table 3

[0141]

[0142] It is detected that the oral keratinized gingiva regeneration repair scaffolds prepared in Examples 1-3 have low cytotoxicity. The use of other modifiers, without secondary modification, the use of other covalent linking agents, the selection of other linking methods, and the like will increase the cytotoxicity.

[0143] Test Example 5

[0144] Antibacterial performance test

[0145] The antibacterial performance of the oral keratinized gingiva regeneration repair scaffolds prepared in Examples 1-7 and Comparative Examples 1-2 is detected according to the standard WS / T 650-2019 “Evaluation method for antibacterial and bacteriostatic effect”.

[0146] Table 4

[0147]

[0148] It is detected that the oral keratinized gingiva regeneration repair scaffolds prepared in Examples 1-3 have strong antibacterial effect on gram-positive bacteria and gram-negative bacteria. The use of other modifiers, without secondary modification, the use of other covalent linking agents, the use of other linking methods, and the like will reduce the antibacterial effect.

[0149] The above merely describes specific embodiments of the present application, but the protection scope of the present application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily conceived by those skilled in the art, and all such changes and replacements fall within the protection scope and disclosure scope of the present application.

Claims

1. A method for preparing an oral keratinized gingiva regenerative repair scaffold, characterized in that, The preparation method comprises: (1) sequentially treating the decellularized matrix with a first modifier, a fixing agent, and a second modifier to obtain an enhanced decellularized matrix; After mixing the carrier solution and the antibacterial natural product solution, an ionic crosslinking agent is added dropwise, and the obtained precipitate after centrifugation is the drug-loaded nanoparticles; (2) mixing the enhanced decellularized matrix, the drug-loaded nanoparticles, and a covalent linking agent, washing with water, and freeze-drying to obtain the product.

2. The method for preparing the oral keratosis gingival regeneration and repair scaffold according to claim 1, characterized in that, The first modifier and the second modifier independently comprise any one or a combination of at least two of threonine, serine, lysine, arginine, glutamic acid, aspartic acid, and cysteine; Preferably, the fixing agent comprises any one or a combination of at least two of glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, dicyclohexyl carbodiimide, genipin, and 1.4 butanediol diglycidyl ether.

3. The method for preparing the oral keratinization gingival regeneration repair scaffold according to claim 1, characterized in that, The mass ratio of the decellularized matrix to the first modifier is (0.5-10):1; Preferably, the mass ratio of the decellularized matrix to the fixing agent is (0.1-10):1; Preferably, the mass ratio of the decellularized matrix to the second modifier is 1:(0.2-5).

4. The method for preparing the oral keratinization gingival regeneration repair scaffold according to claim 1, characterized in that, The temperature of the treatment is independently 4-45 ℃, and the time of a single treatment is 0.5-48 h; Preferably, the number of treatments is independently 1-6 times.

5. The method for preparing the oral keratosis gingival regeneration and repair scaffold according to claim 1, characterized in that, The carrier in the carrier solution comprises chitosan, and the solvent comprises an acetic acid aqueous solution; Preferably, the mass percentage content of acetic acid in the acetic acid aqueous solution is 0.5-3%; Preferably, the concentration of the carrier in the carrier solution is 0.2-5 mg / mL.

6. The method for preparing the oral keratosis gingival regeneration and repair scaffold according to claim 1, characterized in that, The natural antibacterial product comprises any one or a combination of at least two of curcumin, quercetin, and resveratrol; Preferably, the solvent of the natural antibacterial product solution comprises ethanol, glacial acetic acid, or ethyl acetate; Preferably, the volume ratio of the natural antibacterial product solution to the carrier solution is (0.1-5):1; Preferably, the concentration of the natural antibacterial product in the natural antibacterial product solution is 0.2-5 mg / mL.

7. The method for preparing the oral keratinization gingival regeneration repair scaffold according to claim 1, characterized in that, The ionic crosslinking agent comprises a sodium tripolyphosphate aqueous solution; Preferably, the concentration of sodium tripolyphosphate in the sodium tripolyphosphate aqueous solution is 0.2-100 mg / mL; Preferably, the reaction endpoint of adding the ionic crosslinking agent is that opalescence appears and does not disappear; Preferably, the centrifugation is performed at a speed of 8000-12000 rpm for 10-20 min at a temperature of 4-26 ℃; Preferably, the obtained precipitate after centrifugation in step (1) is further washed with a cleaning agent, and the number of washing times is 1-3 times; Preferably, the cleaning agent comprises a methanol aqueous solution with a mass percentage content of 5-20%.

8. The method for preparing the oral keratinization gingival regeneration repair scaffold according to claim 1, characterized in that, The covalent linking agent comprises any one or a combination of at least two of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and dicyclohexyl carbodiimide; Preferably, the ratio of the enhanced decellularized matrix to the covalent linking agent is 1 g:(0.001-0.1) mol. Preferably, the mass ratio of the drug-loaded nanoparticles to the enhanced decellularized matrix is (0.001-0.05):

1.

9. The method for preparing the oral keratinization gingival regeneration repair scaffold according to claim 1, characterized in that, The mixing in step (2) is carried out under solution conditions, and the solvent includes deionized water, PBS buffer, MES buffer, and normal saline. Preferably, the solid-liquid ratio of the mixing in step (2) is 1:(10-500). Preferably, the mixing in step (2) is carried out 1-4 times, the temperature is 4-45 ℃, and the mixing time is 0.5-48 h.

10. An oral keratinized gingiva regenerative repair scaffold prepared by the method according to any one of claims 1-9.

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