A tooth extraction socket filling hemostatic material and preparation method thereof

Abstract

The present invention provides a tooth extraction socket hemostatic filling material and a preparation method thereof, and relates to the technical field of medical biomaterials, wherein the preparation method comprises the following steps: preparing N-hydroxybutyl chitosan; cross-linking N-hydroxybutyl chitosan with aldehydes to obtain cross-linked N-hydroxybutyl chitosan; preparing electrospinning solution with cross-linked N-hydroxybutyl chitosan and hydroxyapatite; electrospinning with the electrospinning solution to obtain an electrospinning membrane; pulverizing the electrospinning membrane to obtain a tooth extraction socket hemostatic filling material. The preparation method of the tooth extraction socket filling hemostatic material provided by the present invention selects a natural polymer material subjected to derivatization treatment, performs gradient weak cross-linking, realizes instantaneous hemostasis, gels after forming a blood clot, and closes the hemostasis; at the same time, hydroxyapatite is entrained by the thermosensitive characteristics of the derived polymer material to stabilize the bone formation space; a nanoscale fiber structure is formed by electrospinning, effectively guiding new bone regeneration, and promoting the formation of alveolar bone in the tooth extraction socket.

Description

Technical Field

[0001] The invention relates to the technical field of medical biomaterials, and in particular to a tooth extraction socket filling hemostatic material and a preparation method thereof. Background Art

[0002] Tooth extraction is the most basic operation in oral and maxillofacial surgery. Like other operations, it will cause varying degrees of damage to local soft and hard tissues, causing bleeding, swelling, pain, infection, maxillary sinus perforation and other symptoms. Improper treatment will cause more serious complications. It is generally believed that a blood clot will form at the extraction socket 15-20 minutes after tooth extraction to seal the wound. The blood clot plays an important role in infiltrating epithelial cells and promoting alveolar bone reconstruction. Removing the blood clot significantly delays the healing of the extraction socket. Therefore, ensuring sufficient and stable blood clots in the extraction socket is crucial for the repair of soft tissue in the extraction socket.

[0003] At present, in addition to non-absorbable materials such as gauze and cotton balls, commonly used absorbable hemostatic materials for extraction sockets include gelatin, oxidized cellulose, fibrin, chitosan, collagen, etc. These hemostatic materials are all absorbable and do not need to be removed a second time, and are highly accepted by patients. However, these hemostatic materials only achieve the effect of compression hemostasis by covering the wound surface, and have a single function. They can neither promote the formation of blood clots nor fix the blood clots after they have coagulated, so that they are not easily destroyed or fall off. They are not suitable for patients with massive bleeding and coagulation disorders in the extraction sockets. At the same time, they will degrade in a short time and cannot seal the extraction wound or fill the extraction socket, which is not conducive to the regeneration of alveolar bone in the extraction socket. Summary of the invention

[0004] The problem solved by the invention is that the existing hemostatic materials for tooth extraction sockets are not conducive to the regeneration of alveolar bone in the tooth extraction sockets.

[0005] In order to solve the above problems, the present invention provides a method for preparing a hemostatic filling material for a tooth extraction socket, comprising the following steps:

[0006] S1: Preparation of N-hydroxybutyl chitosan using chitosan as raw material;

[0007] S2: cross-linking the N-hydroxybutyl chitosan with aldehydes to obtain cross-linked N-hydroxybutyl chitosan;

[0008] S3: preparing an electrospinning solution using the cross-linked N-hydroxybutyl chitosan and hydroxyapatite;

[0009] S4: performing electrospinning with the electrospinning solution to obtain an electrospinning membrane;

[0010] S5: crushing the electrospun membrane to obtain a tooth extraction socket hemostatic filling material.

[0011] Optionally, the chitosan has a deacetylation degree of 80% to 94% and a molecular weight of 5000Da to 2000000Da.

[0012] Optionally, step S1 includes the following steps:

[0013] S11: preparing alkalized chitosan using alkali solution and the chitosan as raw materials;

[0014] S12: adding isopropanol and water to the alkalized chitosan, stirring, adding a phase transfer catalyst, heating in a water bath at 50° C. to 85° C., stirring, to obtain a chitosan pre-catalytic solution;

[0015] S13: adding an etherifying agent to the chitosan pre-catalytic solution, heating to 50° C. to 85° C., reacting to obtain a crude N-hydroxybutyl chitosan product;

[0016] S14: washing the crude N-hydroxybutyl chitosan product with ethanol and adjusting the pH value to 6.5-7.5 to obtain a primary N-hydroxybutyl chitosan product;

[0017] S15: washing and dialyzing the N-hydroxybutyl chitosan primary product, and then vacuum drying at 50° C. to 80° C. to obtain the N-hydroxybutyl chitosan.

[0018] Optionally, step S11 comprises: dissolving NaOH in water, cooling in an ice-water bath to 15° C. to 30° C., adding chitosan thereto, stirring, and filtering to obtain alkalized chitosan.

[0019] Optionally, in step S12, the mass ratio of alkalized chitosan, isopropanol and water is 1:(2-10):(2-15).

[0020] Optionally, the etherifying agent in step S13 is 1,2-butylene oxide.

[0021] Optionally, step S2 includes:

[0022] S21: mixing N-hydroxybutyl chitosan with ethanol, wherein the mass concentration of N-hydroxybutyl chitosan is 1%-30%, to obtain a suspension;

[0023] S22: adding a cross-linking agent to the suspension, stirring and cross-linking, and then filtering to obtain a solidified product;

[0024] S23: After the solidified material is mixed with alcohol and washed, it is filtered and dried to obtain cross-linked N-hydroxybutyl chitosan.

[0025] Optionally, step S3 comprises: dissolving the cross-linked N-hydroxybutyl chitosan in dichloromethane, adding hydroxyapatite, wherein the mass ratio of the cross-linked N-hydroxybutyl chitosan to the hydroxyapatite is (5-20): (0.1-3), stirring, and obtaining an electrospinning solution.

[0026] Optionally, the process conditions of electrospinning in step S4 are: voltage intensity 25 kV, flow rate 1.0 mL / h, receiving material is aluminum foil, and the spinning needle is 5 cm to 10 cm away from the receiving position.

[0027] Another object of the present invention is to provide a tooth extraction socket hemostatic filling material, which is prepared by the method for preparing the tooth extraction socket hemostatic filling material as described above.

[0028] Compared with the prior art, the method for preparing the tooth extraction socket hemostatic material provided by the present invention has the following advantages:

[0029] The preparation method of the hemostatic material for filling a tooth extraction socket provided by the present invention selects a derivatized natural polymer material, performs gradient weak cross-linking, realizes instantaneous hemostasis, forms a blood clot and then gels, and seals the hemostasis; at the same time, the temperature-sensitive property of the derived polymer material is used to entrain hydroxyapatite to stabilize the bone formation space; a nano-scale fiber structure is formed by electrospinning to effectively guide new bone regeneration and promote the formation of alveolar bone in the tooth extraction socket; nano-particles are obtained by mechanical crushing to facilitate the filling of the tooth extraction socket without dead ends. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a scanning electron microscope image of the tooth extraction socket hemostatic filling material prepared in Example 1 of the present invention;

[0031] Figure 2 It is the infrared spectra of chitosan and N-hydroxybutyl chitosan in Example 1 of the present invention. DETAILED DESCRIPTION

[0032] The present invention is now further described in detail. The embodiments described below are exemplary and intended to be used to explain the present invention, but cannot be understood as limiting the present invention. All other embodiments obtained by ordinary technicians in this field without creative work based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0033] Existing hemostatic filling materials, such as patent CN112316209A, an oral hemostatic and repair material and its preparation method, use decalcified bone particles as carriers of oral hemostatic and repair materials in this patent, the collagen structure contained in it has an inductive effect on the mineral deposition of bone in the wound surface, and combined with growth factors and other bone grafting materials, it can effectively initiate and control the mineralization process, promote the formation of new bone, thereby improving the oral hemostatic and repair materials on the regeneration of new bone in the defect site after tooth extraction; this patent cannot fill without dead angles, and there is a problem of rapid degradation; patent CN111849013A, a nano-hydroxyapatite-silk fibroin mineralized scaffold and its preparation method and use, obtained by immersing the silk fibroin scaffold in a mineralizing solution, the hydrophilicity of the mineralized scaffold is significantly improved, the biocompatibility is good, the cell migration and growth can be promoted, and the mechanical properties and good bone differentiation and induction ability are good; although the performance is better than that of traditional nano-hydroxyapatite / silk fibroin composite materials, but The problem is that the scaffold can only provide good filling and guide bone regeneration, but cannot stop bleeding, and needs to be used in conjunction with a hemostatic material; Patent CN110090320B discloses an extraction socket repair scaffold with antibacterial activity and a preparation method thereof. The repair scaffold provided in the patent is a conical structure that can cooperate with the extraction socket, and the conical structure is formed by solidifying the repair material, and a layer of collagen is attached to the bottom of the cone, which is a dense membrane obtained by extrusion after freeze-drying. The repair material includes a collagen-hydroxyapatite complex, collagen, and minocycline hydrochloride; although the repair scaffold can achieve the filling of the extraction socket, the conical structure has strong targeting and poor plasticity, and is not generally applicable; Patent CN112587729A discloses a bone repair material, which is used to provide a bone repair material after defatting and deproteinizing a bovine femur to remove antigens and other substances, and maintain the compressive properties and bone conductivity of the bone repair material; the bone repair material can only guide bone regeneration, and needs to be used in conjunction with a hemostatic material, and the material is in a discrete state in the extraction socket and is not easy to shape.

[0034] In summary, the existing hemostatic filling materials for tooth extraction sockets have the following shortcomings: single function, only able to stop bleeding or repair bones; inconvenient to use, regular shape, poor adaptability to irregular wound surfaces, and failure to ensure filling without dead angles; bone repair materials are easily dispersed, have no shaping function, and are difficult to effectively maintain space.

[0035] In order to solve the problem that the existing extraction socket hemostatic filling materials are not conducive to the regeneration of alveolar bone in the extraction socket, the present invention provides a method for preparing the extraction socket hemostatic filling material, the preparation method comprising the following steps:

[0036] S1: Preparation of N-hydroxybutyl chitosan using chitosan as raw material;

[0037] S2: cross-linking N-hydroxybutyl chitosan with aldehydes to obtain cross-linked N-hydroxybutyl chitosan;

[0038] S3: Preparation of electrospinning solution by cross-linking N-hydroxybutyl chitosan and hydroxyapatite;

[0039] S4: performing electrospinning with an electrospinning solution to obtain an electrospinning membrane;

[0040] S5: crushing the electrospun membrane to obtain a hemostatic filling material for a tooth extraction socket.

[0041] The molecular structure of chitosan contains a large number of active functional groups, such as hydroxyl, amino, acetylamino, etc. The present invention selectively chemically modifies chitosan to prepare a new chitosan derivative, thereby preparing a new extraction socket hemostatic filling material; hydroxyapatite is the main inorganic component of human and animal bones, promotes bone growth in the extraction socket, realizes chemical bonding with body tissues at the interface, has a certain solubility in the body, can release ions that are harmless to the body, participates in body metabolism, has a stimulating or inducing effect on bone hyperplasia, can promote the repair of defective tissues, and shows biological activity.

[0042] Specifically, the present application is to improve the hemostatic function, as shown in the following formula:

[0043]

[0044] First, hydroxybutyl groups are introduced by substitution on the C2-NH2 of chitosan to obtain N-hydroxybutyl chitosan rich in hydroxybutyl, and then cross-linked with aldehydes to maintain the properties of chitosan while making it thermosensitive, so that the material can instantly absorb blood, form a blood clot and then gel, forming a gel to seal and stop bleeding, and at the same time, it forms a stable gel with thermosensitivity, which entrains hydroxyapatite to form a stable bone formation space and maintain the good shape of the extraction socket; a nano-scale fiber structure is formed by electrospinning, which effectively guides the regeneration of new bone and promotes the formation of alveolar bone in the extraction socket; nano-fiber particles are obtained by mechanical crushing, which not only helps to increase the contact area between the material and the wound and improve the liquid absorption efficiency, but also can maintain the filling and sealing state, achieve filling without dead angles, and facilitate the filling of the extraction socket.

[0045] The preparation method of the hemostatic material for filling a tooth extraction socket provided by the present invention selects a derivatized natural polymer material, performs gradient weak cross-linking, realizes instantaneous hemostasis, forms a blood clot and then gels, and seals the hemostasis; at the same time, the temperature-sensitive property of the derived polymer material is used to entrain hydroxyapatite to stabilize the bone formation space; a nano-scale fiber structure is formed by electrospinning to effectively guide new bone regeneration and promote the formation of alveolar bone in the tooth extraction socket; nano-particles are obtained by mechanical crushing to facilitate the filling of the tooth extraction socket without dead ends.

[0046] In order to ensure the performance of the hemostatic filling material for the tooth extraction socket, the present application preferably adopts a chitosan with a deacetylation degree of 80% to 94% and a molecular weight of 5000Da to 2000000Da.

[0047] The preferred step S1 of the present application includes the following steps:

[0048] S11: preparing alkalized chitosan using alkali solution and chitosan as raw materials;

[0049] S12: adding isopropanol and water to the alkalized chitosan, stirring, adding a phase transfer catalyst, heating in a water bath at 50° C. to 85° C., stirring, to obtain a chitosan pre-catalytic solution;

[0050] S13: adding an etherifying agent to the chitosan pre-catalytic solution, heating to 50° C. to 80° C., reacting to obtain a crude N-hydroxybutyl chitosan product;

[0051] S14: washing the crude N-hydroxybutyl chitosan product with ethanol, and adjusting the pH value to 6.5-7.5 to obtain a primary N-hydroxybutyl chitosan product;

[0052] S15: washing and dialyzing the initial N-hydroxybutyl chitosan product, and then vacuum drying at 50° C. to 80° C. to obtain N-hydroxybutyl chitosan.

[0053] Specifically, the preferred step S11 comprises: dissolving NaOH in water, cooling in an ice-water bath to 15°C to 30°C, adding chitosan thereto, stirring sufficiently, preferably for 3 to 12 hours, fluffing the chitosan, and filtering to obtain alkalized chitosan; preferably, the mass ratio of NaOH, water and chitosan in this step is 1:1:1.

[0054] The chitosan is further pre-catalyzed. Specifically, isopropanol and water are added to the alkalized chitosan. Preferably, the mass ratio of the alkalized chitosan, isopropanol and water in step S12 is 1:(2-10):(2-15). The chitosan is stirred until the alkalized chitosan is uniformly dispersed. A phase transfer catalyst is added thereto. Preferably, the phase transfer catalyst is sodium dodecyl sulfate. The mixture is heated in a water bath at 50°C to 85°C and stirred sufficiently, preferably for 3 to 18 hours, to obtain a chitosan pre-catalytic solution.

[0055] To further prepare hydroxyalkylated chitosan, an etherifying agent is added to the chitosan pre-catalytic solution, preferably the etherifying agent in step S13 is 1,2-butylene oxide, and the etherifying agent is preferably added dropwise at a dropping speed of 20-80 drops / s, the temperature is raised to 50°C to 80°C, an etherification reaction is carried out, the reaction time is controlled to 12h-36h, a hydroxybutyl group is introduced on C2-NH2, and a crude N-hydroxybutyl chitosan product is obtained.

[0056] To terminate the reaction, the crude N-hydroxybutyl chitosan product is washed with ethanol (preferably 75% to 100% by mass), and the pH is adjusted to 6.5-7.5 with 0.1 mol / L to 1 mol / L hydrochloric acid to obtain the initial N-hydroxybutyl chitosan product.

[0057] The product is purified. The preferred product of the present application is centrifuged and washed 3-5 times with anhydrous ethanol or acetone, dialyzed in deionized water for 24h-72h using a dialysis bag with a molecular weight cutoff of 3000. After the dialysis, the product is vacuum dried at a temperature of 50-80°C to obtain N-hydroxybutyl chitosan.

[0058] The present application further weakly cross-links the hydroxyl groups of N-hydroxybutyl chitosan with aldehydes to achieve instantaneous blood absorption and stabilize blood clots, so that the material is fully gelled and sealed to stop bleeding. In addition, the thermosensitivity of N-hydroxybutyl chitosan is used to entrain nano-hydroxyapatite to form a stable bone formation space; specifically, the preferred step S2 of the present application includes:

[0059] S21: mixing N-hydroxybutyl chitosan with ethanol, wherein the mass concentration of N-hydroxybutyl chitosan is 1%-30%, to obtain a suspension;

[0060] S22: adding a crosslinking agent to the suspension, stirring and crosslinking, and then filtering to obtain a solidified product; preferably, the crosslinking agent is glyoxal, and the stirring and crosslinking time is preferably 1 h to 6 h;

[0061] S23: After the solidified product is mixed with alcohol and washed, it is filtered and dried to obtain cross-linked N-hydroxybutyl chitosan; preferably, the solidified product is mixed with alcohol in a ratio of 1:1-5 and washed 2-5 times to adsorb excess cross-linking agent, and after washing, the washing liquid is removed by filtering, and the product is dried at 30-55°C or vacuum dried to obtain cross-linked N-hydroxybutyl chitosan.

[0062] To promote alveolar bone regeneration, the preferred step S3 of the present application includes: dissolving cross-linked N-hydroxybutyl chitosan in dichloromethane, adding hydroxyapatite, the mass ratio of cross-linked N-hydroxybutyl chitosan to hydroxyapatite is (5-20): (0.1-3), stirring, and obtaining an electrospinning solution.

[0063] The preferred process conditions for electrospinning in step S4 of the present application are: voltage intensity 25 kV, flow rate 1.0 mL / h, receiving material is aluminum foil, and the spinning needle is 5 cm to 10 cm away from the receiving position.

[0064] The nanofiber structure formed by electrospinning in the present application can effectively guide new bone regeneration and promote the formation of alveolar bone in the tooth extraction socket.

[0065] Another object of the present invention is to provide a hemostatic filling material for a tooth extraction socket, wherein the filling material is prepared by the method for preparing a hemostatic filling material for a tooth extraction socket as described above.

[0066] The extraction socket hemostatic filling material provided by the present invention selects a derivatized natural polymer material, performs gradient weak cross-linking, realizes instantaneous hemostasis, forms a blood clot and then gels, and seals the hemostasis; at the same time, the temperature-sensitive property of the derived polymer material is used to entrain hydroxyapatite to stabilize the bone formation space; a nano-scale fiber structure is formed by electrospinning, which effectively guides new bone regeneration and promotes the formation of alveolar bone in the extraction socket; nano-particles are obtained by mechanical crushing, which is convenient for filling the extraction socket without dead angles.

[0067] Specifically, the present application introduces hydroxybutyl groups by substitution on the C2-NH2 of chitosan to obtain N-hydroxybutyl chitosan rich in hydroxybutyl, and weakly cross-links the hydroxyl groups of N-hydroxybutyl chitosan with aldehydes to achieve instantaneous blood absorption, while stabilizing blood clots and allowing the material to fully gel and seal for hemostasis. In addition, the temperature sensitivity of N-hydroxybutyl chitosan is used to entrain nano-hydroxyapatite to form a stable bone formation space; in terms of practicality, an electrospinning process is used to obtain a nano-microwire structure that can guide bone regeneration. After mechanical crushing, the contact area is increased, so that the material can be arbitrarily shaped to meet the performance of filling without dead ends.

[0068] The hemostatic material for filling the tooth extraction socket provided by the present invention can not only stop bleeding, but also has the function of guiding bone regeneration; the hemostatic effect not only blocks the wound to achieve the effect of exogenous hemostasis, but also can aggregate effective components such as platelets and leukocytes in the blood, promote the formation of blood clots at the injured site, and stimulate endogenous hemostasis; the blood clot and the hemostatic material form a stable structure at the wound site, achieve the effect of rapid coagulation, stable blood clots, and protection of the tooth extraction socket wound; at the same time, it can promote cell growth, promote osteoblast differentiation and the formation of trabeculae, thereby achieving tooth extraction socket filling and preventing gingival atrophy at the tooth extraction site, and is an ideal hemostatic filling material for tooth extraction sockets.

[0069] Example 1

[0070] S11: dissolving NaOH in an equal amount of purified water, cooling to 20°C in an ice water bath, adding an equal amount of chitosan, stirring for 5 hours, and fluffing the chitosan to obtain alkaline chitosan; wherein the chitosan has a deacetylation degree of 85% and a molecular weight of 1,200,000 Da;

[0071] S12: add 10 g of isopropanol and 40 g of purified water (alkaline chitosan: isopropanol: purified water = 1:2:8) to 5 g of alkalized chitosan, heat in a water bath at 60°C, stir thoroughly for 5 h, then add 0.1 g of sodium dodecyl sulfate, continue stirring for 3 h, and obtain a chitosan pre-catalytic solution;

[0072] S13: 100 mL of 1,2-butylene oxide was added dropwise to the chitosan pre-catalytic solution at a dropping speed of 50 drops / s, and the etherification reaction was carried out at 60° C. for 12 h to obtain a crude N-hydroxybutyl chitosan product;

[0073] S14: washing the crude N-hydroxybutyl chitosan product with anhydrous ethanol, and then adjusting the pH to 7 with a 0.5 M hydrochloric acid solution to obtain a primary N-hydroxybutyl chitosan product;

[0074] S15: The initial product of N-hydroxybutyl chitosan was washed again with anhydrous ethanol for three times, and dialyzed in deionized water for 48 h using a dialysis bag with a molecular weight cutoff of 3000. After the dialysis, the product was vacuum dried at 50° C. to obtain N-hydroxybutyl chitosan;

[0075] S21: dispersing N-hydroxybutyl chitosan in anhydrous ethanol, wherein the mass concentration of N-hydroxybutyl chitosan is 15%, and stirring for 30 minutes to obtain a suspension;

[0076] S22: adding 0.03% by mass of glyoxal to the suspension, stirring and cross-linking for 3 hours, and filtering to obtain a solid;

[0077] S23: adding ethanol 3 times the weight of the solidified product to wash 3 times to remove excess cross-linking agent, filtering to remove anhydrous ethanol after washing, and vacuum drying at 50° C. to obtain cross-linked N-hydroxybutyl chitosan;

[0078] S3: dissolving cross-linked N-hydroxybutyl chitosan in 50 mL of dichloromethane, adding 1 g of hydroxyapatite and stirring to obtain an electrospinning solution;

[0079] S4: electrospinning with the electrospinning solution, adjusting the voltage and spinning distance, electrospinning at a voltage intensity of 25 kV and a flow rate of 1.0 mL / h, and receiving with aluminum foil to obtain an electrospinning membrane;

[0080] S5: vacuum drying the electrospun membrane at 35° C., mechanically crushing the electrospun membrane to obtain nanofiber particles, and obtaining a tooth extraction socket hemostatic filling material.

[0081] The scanning electron microscope image of the hemostatic filling material for tooth extraction socket prepared in this embodiment is shown in FIG. Figure 1 shown.

[0082] Example 2

[0083] S11: dissolving NaOH in an equal amount of purified water, cooling to 15°C in an ice water bath, adding an equal amount of chitosan, stirring for 3 hours, and fluffing the chitosan to obtain alkaline chitosan; wherein the chitosan has a deacetylation degree of 85% and a molecular weight of 50,000 Da;

[0084] S12: Add 25 g of isopropanol and 60 g of purified water (alkaline chitosan: isopropanol: purified water = 1:5:12) to 5 g of alkalized chitosan, heat in a water bath at 75°C, stir thoroughly for 2 h, then add 0.1 g of sodium dodecyl sulfate, continue stirring for 5 h, and obtain a chitosan pre-catalytic solution;

[0085] S13: 80 mL of 1,2-butylene oxide was added dropwise to the chitosan pre-catalytic solution at a dropping speed of 20 drops / s, and the etherification reaction was carried out at 80° C. for 15 h to obtain a crude N-hydroxybutyl chitosan product;

[0086] S14: washing the crude N-hydroxybutyl chitosan product with 95% ethanol, and then adjusting the pH to 7 with a 0.5 M hydrochloric acid solution to obtain a primary N-hydroxybutyl chitosan product;

[0087] S15: The initial product of N-hydroxybutyl chitosan was washed again with anhydrous ethanol for three times, and dialyzed in deionized water for 65 h using a dialysis bag with a molecular weight cutoff of 3000. After the dialysis, the product was vacuum dried at 70°C to obtain N-hydroxybutyl chitosan;

[0088] S21: dispersing N-hydroxybutyl chitosan in anhydrous ethanol, wherein the mass concentration of N-hydroxybutyl chitosan is 15%, and stirring for 50 minutes to obtain a suspension;

[0089] S22: adding 0.05% by mass of glyoxal to the suspension, stirring and cross-linking for 2 h, and filtering to obtain a solidified product;

[0090] S23: adding 5 times the weight of ethanol to the solidified product for washing three times to remove excess cross-linking agent, filtering to remove anhydrous ethanol after washing, and vacuum drying at 50° C. to obtain cross-linked N-hydroxybutyl chitosan;

[0091] S3: dissolving cross-linked N-hydroxybutyl chitosan in 50 mL of dichloromethane, adding 1 g of hydroxyapatite and stirring to obtain an electrospinning solution;

[0092] S4: electrospinning with the electrospinning solution, adjusting the voltage and spinning distance, electrospinning at a voltage intensity of 25 kV and a flow rate of 1.0 mL / h, and receiving with aluminum foil to obtain an electrospinning membrane;

[0093] S5: vacuum drying the electrospun membrane at 40° C., mechanically crushing the electrospun membrane to obtain nanofiber particles, and obtaining a tooth extraction socket hemostatic filling material.

[0094] Example 3

[0095] S11: dissolving NaOH in an equal amount of purified water, cooling to 30° C. in an ice water bath, adding an equal amount of chitosan, stirring for 12 hours, and fluffing the chitosan to obtain alkaline chitosan; wherein the chitosan has a deacetylation degree of 90% and a molecular weight of 300,000 Da;

[0096] S12: Add 50 g of isopropanol and 10 g of purified water (alkaline chitosan: isopropanol: purified water = 1:10:2) to 5 g of alkalized chitosan, heat in a 50°C water bath, stir thoroughly for 2 h, then add 0.1 g of sodium dodecyl sulfate, continue stirring for 18 h, and obtain a chitosan pre-catalytic solution;

[0097] S13: 80 mL of 1,2-butylene oxide was added dropwise to the chitosan pre-catalytic solution at a dropping speed of 80 drops / s, and the etherification reaction was carried out at 50° C. for 36 h to obtain a crude N-hydroxybutyl chitosan product;

[0098] S14: washing the crude N-hydroxybutyl chitosan product with 95% ethanol, and then adjusting the pH to 7 with a 0.5 M hydrochloric acid solution to obtain a primary N-hydroxybutyl chitosan product;

[0099] S15: The initial product of N-hydroxybutyl chitosan was washed again with anhydrous ethanol for three times, and dialyzed in deionized water for 12 h using a dialysis bag with a molecular weight cutoff of 3000. After the dialysis, the product was vacuum dried at 80° C. to obtain N-hydroxybutyl chitosan;

[0100] S21: dispersing N-hydroxybutyl chitosan in anhydrous ethanol, wherein the mass concentration of N-hydroxybutyl chitosan is 15%, and stirring for 50 minutes to obtain a suspension;

[0101] S22: adding 0.05% by mass of glyoxal to the suspension, stirring and cross-linking for 2 h, and filtering to obtain a solidified product;

[0102] S23: adding 5 times the weight of ethanol to the solidified product for washing three times to remove excess cross-linking agent, filtering to remove anhydrous ethanol after washing, and vacuum drying at 50° C. to obtain cross-linked N-hydroxybutyl chitosan;

[0103] S3: dissolving cross-linked N-hydroxybutyl chitosan in 40 mL of dichloromethane, adding 0.5 g of hydroxyapatite and stirring to obtain an electrospinning solution;

[0104] S4: electrospinning with the electrospinning solution, adjusting the voltage and spinning distance, electrospinning at a voltage intensity of 25 kV and a flow rate of 1.0 mL / h, and receiving with aluminum foil to obtain an electrospinning membrane;

[0105] S5: vacuum drying the electrospun membrane at 40° C., mechanically crushing the electrospun membrane to obtain nanofiber particles, and obtaining a tooth extraction socket hemostatic filling material.

[0106] Comparative Example 1

[0107] S1: Dissolve chitosan in 50 mL of dichloromethane, add 1 g of hydroxyapatite and stir to obtain an electrospinning solution;

[0108] S4: electrospinning with the electrospinning solution, adjusting the voltage and spinning distance, electrospinning at a voltage intensity of 25 kV and a flow rate of 1.0 mL / h, and receiving with aluminum foil to obtain an electrospinning membrane;

[0109] S5: vacuum drying the electrospun membrane at 35° C., mechanically crushing the electrospun membrane to obtain nanofiber particles, and obtaining a tooth extraction socket hemostatic filling material.

[0110] Comparative Example 2

[0111] S11: dissolving NaOH in an equal amount of purified water, cooling to 20°C in an ice water bath, adding an equal amount of chitosan, stirring for 5 hours, and fluffing the chitosan to obtain alkaline chitosan; wherein the chitosan has a deacetylation degree of 85% and a molecular weight of 1,200,000 Da;

[0112] S12: add 10 g of isopropanol and 40 g of purified water (alkaline chitosan: isopropanol: purified water = 1:2:8) to 5 g of alkalized chitosan, heat in a water bath at 60°C, stir thoroughly for 5 h, then add 0.1 g of sodium dodecyl sulfate, continue stirring for 3 h, and obtain a chitosan pre-catalytic solution;

[0113] S13: 100 mL of 1,2-butylene oxide was added dropwise to the chitosan pre-catalytic solution at a dropping speed of 50 drops / s, and the etherification reaction was carried out at 60° C. for 12 h to obtain a crude N-hydroxybutyl chitosan product;

[0114] S14: washing the crude N-hydroxybutyl chitosan product with anhydrous ethanol, and then adjusting the pH to 7 with a 0.5 M hydrochloric acid solution to obtain a primary N-hydroxybutyl chitosan product;

[0115] S15: The initial product of N-hydroxybutyl chitosan was washed again with anhydrous ethanol for three times, and dialyzed in deionized water for 48 h using a dialysis bag with a molecular weight cutoff of 3000. After the dialysis, the product was vacuum dried at 50° C. to obtain N-hydroxybutyl chitosan;

[0116] S21: dispersing N-hydroxybutyl chitosan in anhydrous ethanol, wherein the mass concentration of N-hydroxybutyl chitosan is 15%, and stirring for 30 minutes to obtain a suspension;

[0117] S22: adding 0.03% by mass of glyoxal to the suspension, stirring and cross-linking for 3 hours, and filtering to obtain a solid;

[0118] S23: adding ethanol 3 times the weight of the solidified product to wash 3 times to remove excess cross-linking agent, filtering to remove anhydrous ethanol after washing, and vacuum drying at 50° C. to obtain cross-linked N-hydroxybutyl chitosan;

[0119] S3: dissolving the cross-linked N-hydroxybutyl chitosan in 50 mL of dichloromethane to obtain an electrospinning solution;

[0120] S4: electrospinning with the electrospinning solution, adjusting the voltage and spinning distance, electrospinning at a voltage intensity of 25 kV and a flow rate of 1.0 mL / h, and receiving with aluminum foil to obtain an electrospinning membrane;

[0121] S5: vacuum drying the electrospun membrane at 35° C., mechanically crushing the electrospun membrane to obtain nanofiber particles, and obtaining a tooth extraction socket hemostatic filling material.

[0122] Test Example 1

[0123] The liquid absorption rate, liquid absorption time, elastic modulus, and viscosity modulus of the hemostatic materials of each embodiment and comparative example were tested and compared, and the test data are shown in Table 1:

[0124] Table 1

[0125]

[0126]

[0127] It can be seen from the above data that the hemostatic filling materials prepared in each embodiment of the present invention have good liquid absorption performance; through the elastic modulus and the viscous modulus, it can be seen that the gel formed after the material stops bleeding firmly fills the tooth extraction socket, thereby proving that the hemostatic filling material provided by the present invention can not only absorb blood instantly to form a gel to seal and stop bleeding, but also form a stable gel in a temperature-sensitive manner to entrain hydroxyapatite to form a stable bone formation space.

[0128] Compared with Example 1, the hemostatic filling material provided in Comparative Example 1 does not chemically modify chitosan. The prepared hemostatic filling material only maintains the liquid absorption performance of chitosan itself, has no support and cannot play the function of sealing the tooth extraction socket.

[0129] The hemostatic filling material provided in Comparative Example 2 does not contain hydroxyapatite compared to Example 1. The prepared hemostatic filling material has no significant difference in fluid absorption capacity, support and blocking effect. However, without the addition of hydroxyapatite, it cannot play a role in guiding bone regeneration.

[0130] Test Example 2: Cytotoxicity Test

[0131] The extracts of each embodiment, comparative example, negative control (polyethylene, 1 mL of cell culture medium was added to every 6 cm2 of negative control, and extracted at 60 rpm and 37°C for 24 h), positive control (10% DMSO solution, prepared freshly) and medium control were placed in the wells of L929 mouse fibroblast (MEF) cell culture plates and cultured in a 5% CO2, 37°C cell culture medium. The MTT method was used for evaluation. The results are shown in Table 2.

[0132] Under the conditions of this experiment, the cell survival rate of the test sample group exceeded 90%, the test sample group had low cytotoxicity and no potential cytotoxicity.

[0133] Table 2

[0134]

[0135]

[0136] Test Example 3: Infrared Characterization

[0137] Chitosan and the N-hydroxybutyl chitosan samples of Example 1 were ground and made into thin film discs by KBr tableting method. The samples were analyzed by Fourier transform infrared spectrometer in the wavelength range of 400-4000cm-1 to obtain infrared spectra. Figure 2 shown.

[0138] from Figure 2 It can be seen that after chitosan modification, 3363 cm -1 The strong peak at 2890 cm-1 is still retained, which is attributed to the superposition of the stretching vibration absorption peaks of -OH and NH. -1 and 1461cm -1 There are obvious new absorption peaks at 1000-1200cm, which are the stretching vibration and asymmetric deformation of CH, indicating that -CH3 and -CH2 are added after modification, that is, hydroxybutyl groups are introduced. -1 There is no significant change in the absorption peak within the range, which is because C6-OH and C4-OH are not replaced, and the absorption peak is not significantly enhanced or weakened. The absorption peak of chitosan is 1461cm -1 The CN absorption peak at changed, indicating that the reaction occurred on C2-NH2.

[0139] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for preparing a hemostatic filling material for a tooth extraction socket, characterized in that: The steps include: S1: Preparation of N-hydroxybutyl chitosan using chitosan as raw material; S2: cross-linking the N-hydroxybutyl chitosan with aldehydes to obtain cross-linked N-hydroxybutyl chitosan; Step S2 includes: S21: mixing N-hydroxybutyl chitosan with ethanol, wherein the mass concentration of N-hydroxybutyl chitosan is 1%-30%, to obtain a suspension; S22: adding a cross-linking agent to the suspension, stirring and cross-linking, and then filtering to obtain a solidified product; S23: mixing the solidified product with alcohol, washing it, filtering it with suction, and drying it to obtain cross-linked N-hydroxybutyl chitosan; S3: preparing an electrospinning solution using the cross-linked N-hydroxybutyl chitosan and hydroxyapatite; S4: performing electrospinning with the electrospinning solution to obtain an electrospinning membrane; S5: crushing the electrospun membrane to obtain a tooth extraction socket hemostatic filling material.

2. The method for preparing the hemostatic filling material for tooth extraction socket according to claim 1, characterized in that: The deacetylation degree of the chitosan is 80% to 94%, and the molecular weight is 5000Da to 2000000Da.

3. The method for preparing the hemostatic filling material for tooth extraction socket according to claim 1, characterized in that: Step S1 includes the following steps: S11: preparing alkalized chitosan using alkali solution and the chitosan as raw materials; S12: adding isopropanol and water to the alkalized chitosan, stirring, adding a phase transfer catalyst, heating in a water bath at 50° C. to 85° C., stirring, to obtain a chitosan pre-catalytic solution; S13: adding an etherifying agent to the chitosan pre-catalytic solution, heating to 50° C. to 80° C., reacting to obtain a crude N-hydroxybutyl chitosan product; S14: washing the crude N-hydroxybutyl chitosan product with ethanol and adjusting the pH value to 6.5-7.5 to obtain a primary N-hydroxybutyl chitosan product; S15: washing and dialyzing the N-hydroxybutyl chitosan primary product, and then vacuum drying at 50° C. to 80° C. to obtain the N-hydroxybutyl chitosan.

4. The method for preparing the hemostatic filling material for tooth extraction sockets according to claim 3, characterized in that: Step S11 comprises: dissolving NaOH in water, cooling in an ice-water bath to 15° C. to 30° C., adding chitosan thereto, stirring, and filtering to obtain alkalized chitosan.

5. The method for preparing the hemostatic filling material for tooth extraction socket according to claim 3, characterized in that: In step S12, the mass ratio of alkalized chitosan, isopropanol and water is 1:(2-10):(2-15).

6. The method for preparing the hemostatic filling material for tooth extraction sockets according to claim 3, characterized in that: The etherifying agent in step S13 is 1,2-butylene oxide.

7. The method for preparing the hemostatic filling material for tooth extraction sockets according to claim 1, characterized in that: Step S3 comprises: dissolving the cross-linked N-hydroxybutyl chitosan in dichloromethane, adding hydroxyapatite, wherein the mass ratio of the cross-linked N-hydroxybutyl chitosan to the hydroxyapatite is (5-20): (0.1-3), stirring, and obtaining an electrospinning solution.

8. The method for preparing the hemostatic filling material for tooth extraction sockets according to claim 1, characterized in that: The process conditions of electrospinning in step S4 are: voltage intensity 25 kV, flow rate 1.0 mL / h, receiving material is aluminum foil, and the spinning needle is 5 cm to 10 cm away from the receiving position.

9. A hemostatic filling material for tooth extraction sockets, characterized in that: The hemostatic filling material for tooth extraction socket is prepared by the preparation method of any one of claims 1 to 8.

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