A multifunctional composite guided bone regeneration membrane and its preparation and application

Abstract

The present invention relates to the technical field of guided bone regeneration, and in particular to a multifunctional composite guided bone regeneration membrane and its preparation and application. The present invention first grafts amino-modified basic copper phosphate with activated carboxyl groups on poly-L-glutamic acid, and then chemically cross-links with chitosan solution, and prepares a CP / PLGA / CS dense layer with antibacterial and barrier effects by a solvent casting method; then, amino-modified strontium replaces hydroxyapatite and grafts it with carboxyl groups on silk fibroin, and then electrostatically interacts with NH2 on CS, and prepares a Sr-HA / SF / CS porous membrane by freeze-drying. The CP / PLGA / CS dense layer in the multifunctional composite guided bone regeneration membrane of the present invention can sustainably release Cu 2+ , giving the membrane antibacterial function; the biomimetic cytoplasmic matrix of the Sr-HA / SF / CS porous membrane provides physical space for the infiltration and growth of cells, while the continuous release of Sr 2+ and Ca 2+ Both endowed the guided bone regeneration membrane with osteogenic properties.

Description

Technical Field

[0001] The present invention relates to the technical field of guided bone regeneration, and in particular to a multifunctional composite guided bone regeneration membrane and its preparation and application. Background Art

[0002] Bone defects or insufficient bone mass are the key and difficult problems faced by tissue engineering in clinical practice and are also the main cause of implant failure. Guided bone tissue regeneration technology is currently the main means to achieve bone tissue regeneration.

[0003] Guided bone regeneration (GBR) is an effective technology that can produce new bone in areas where the size (width or height) or bone volume is insufficient. The principle of GBR is to form an isolated space with a barrier membrane and make room for a bone substitute for new bone growth. This is because the newly formed bone can be invaded by the epithelial cells of the soft tissue and forced to collapse. However, combining the barrier membrane with the bone substitute during surgery remains a clinical challenge. Guided bone tissue regeneration technology uses membrane materials as a barrier, placed on the surface of the bone defect, to prevent the faster-growing fibrous connective tissue from occupying the space of the bone defect, providing growth space for bone tissue regeneration and reconstruction. Since the two sides of the guided bone regeneration membrane face the epithelial tissue and bone tissue respectively, the two sides of the membrane are given different structures - a dense structure facing the epithelial tissue and a porous structure facing the bone tissue, that is, an asymmetric membrane, which enables it to have both tissue barrier function and guided bone regeneration ability.

[0004] Bone tissue reconstruction mainly includes natural transplant tissue and synthetic biomaterials. Hard tissue replacement materials for defect sites can be divided into natural transplants (autologous grafts, allografts and xenografts) and synthetic materials. These materials are used because they have osteogenic, inductive and / or osteoconductive properties. Ideally, the graft should be biocompatible, easy to mold and / or sculpt, integrate well with natural bone, have sufficient mechanical properties and an ideal replacement rate, and be predictable and well accepted by patients. The principle of membrane technology for bone regeneration is to separate different tissues by surgically placing a physical barrier. The renewal rate of soft tissue is faster than that of bone tissue formation, and the use of a barrier membrane can maintain the defect space to regenerate tissue, otherwise these bone defect areas will be infiltrated and occupied by epithelial cells. Used in conjunction with bone grafts, membranes help stabilize, contain and preserve graft materials.

[0005] Biomedical polymer materials offer advantages such as well-designed structures, excellent bioactivity, stable physical properties, wide availability, and low cost. Polymers have always been a hot topic in the development of GBR applications, with a growing trend year by year. GBR membranes should possess biocompatibility, bioactivity, and tissue selectivity. Soft tissues, such as epithelial and connective tissue, proliferate and migrate relatively rapidly. GBR is designed to isolate soft tissue from bone defects through the membrane, providing a relatively closed environment for tissue growth. Regenerative cells in the bone defect area are maximized to proliferate and differentiate, ensuring preferential osteogenesis and promoting new bone formation.

[0006] During the GBR process, different types of barrier membranes can be applied to bone defects and play a role in osteogenesis. The mechanical properties of several common barrier membranes show that single-component polymers often have lower mechanical properties than composite polymers. Both mechanical and biological properties affect the stability and effectiveness of barrier membranes. Materials used to prepare GBRs are primarily divided into natural polymers (such as collagen, chitosan, silk fibroin, and alginate) and synthetic polymers (such as polylactic-coglycolic acid, polycaprolactone, polyethylene glycol, and polyglycolic acid).

[0007] Functional membranes have received increasing attention in recent years. There are three main preparation methods: (1) loading antibacterial materials to reduce the repair failure rate caused by inflammation; (2) loading bioactive factors to increase bone mass through self-osteogenesis and synergistic osteogenesis; (3) manufacturing of multilayer structures to meet the different requirements of different contact surfaces such as implant surface, bone surface and epithelial tissue in bone regeneration. According to the preparation method, the following three functional membranes are obtained: (1) antibacterial barrier membrane, when loaded with antibiotics, growth factors and adhesion factors, the synthetic membrane can be used as a delivery device for specific preparations. In order to ensure the effectiveness of guided bone regeneration, it is necessary to reduce the inflammatory response caused by bacterial invasion. (2) bioactive barrier membrane, which controls various growth factors with different bioactivities and plays a role in different stages of bone healing, mimicking the natural osteogenesis process. The most important factors currently proven to be osteogenic factors and angiogenic factors. (3) structural layered barrier membrane. The design of multilayer barrier membranes aims to meet more complex requirements and give the barrier membrane richer properties. Each layer should have different biological properties.

[0008] The human oral cavity temperature is approximately 37°C, and the moist cavity environment provides an optimal environment for the growth and reproduction of bacteria, fungi, and viruses. This disrupts the original ecological balance of the oral cavity. This environment makes implanted materials susceptible to bacterial invasion, which in turn affects cell growth and ultimately reduces the repair effect of the alveolar bone. Therefore, while ensuring the functionality of the membrane, it is also necessary to increase the membrane's antibacterial properties. Incorporating metal ions or introducing antibacterial drugs is the most direct way to impart antibacterial properties to the material. Studies have shown that copper deficiency can lead to decreased bone strength in rats. In addition, copper was the first metal antibacterial agent to be certified by the U.S. Environmental Protection Agency (EPA) in 2008. It is reported that more than 300 copper and its compound surfaces have shown antibacterial properties against different bacterial species. These antibacterial copper products claim to kill 99.9% of pathogenic bacteria within 2 hours.

[0009] In summary, although there have been studies on guided bone regeneration with antibacterial properties and cell growth promotion, they are more of a burst release effect and cannot continuously release metal ions to achieve the antibacterial effect. The amino-modified basic copper phosphate and strontium-substituted hydroxyapatite in the present invention can interact with the carboxyl groups on PLGA and SF to achieve a sustained release effect, solving the burst release problem, and the guided regeneration membrane has both antibacterial and osteogenesis effects. Summary of the Invention

[0010] In order to solve the problems existing in the current guided bone regeneration membranes, such as the uncontrollable release of inorganic ions and the inability to achieve continuous antibacterial effects, the purpose of the present invention is to provide a multifunctional composite guided bone regeneration membrane and its preparation and application. The present invention first constructs a dense layer by a solvent casting method, and then constructs a porous layer on the basis of the dense layer by freeze-drying technology, and finally obtains a double-layer composite guided bone regeneration membrane. The multifunctional composite guided bone regeneration membrane prepared by the present invention has a dense layer blended with basic copper phosphate nanoparticles, which has antibacterial function, while increasing hydrophobicity to more effectively prevent the invasion of epithelial cells; the porous layer has a pore structure that simulates the extracellular matrix, and can controllably release Sr 2+ and Ca 2+ Can accelerate the repair of bone defects.

[0011] The multifunctional composite guided bone regeneration membrane (also referred to as the "guided bone regeneration membrane") of the present invention comprises a dense layer and a porous layer, exhibiting excellent biocompatibility. The dense layer is prepared by amidation of poly-L-glutamic acid (PLGA) and chitosan (CS) in equal proportions. Amino-modified basic copper phosphate nanoparticles (CP-NH2) are incorporated into the membrane, and the copper ions have an antibacterial effect. The porous layer is a mixture of silk fibroin (SF) and CS in equal volumes, into which amino-modified strontium-substituted hydroxyapatite (Sr-HA-NH2) is incorporated. Sr can enhance pre-osteoblast differentiation and inhibit osteoclast formation (promoting osteogenesis).

[0012] The purpose of the present invention can be achieved by the following technical solutions:

[0013] The first object of the present invention is to provide a multifunctional composite guided bone regeneration membrane, which is composed of a dense layer and a porous layer; the porous layer is placed on the upper surface of the dense layer;

[0014] The dense layer is obtained by amidating chitosan and poly-L-glutamic acid and then doping amino-modified basic copper phosphate nanoparticles;

[0015] The porous layer is obtained by doping a mixture of silk fibroin and chitosan with amino-modified strontium-substituted hydroxyapatite.

[0016] A second object of the present invention is to provide a method for preparing a multifunctional composite guided bone regeneration membrane, comprising the following steps:

[0017] (S1) adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) to a poly-L-glutamic acid (PLGA) solution, and then adding N-hydroxysuccinimide (NHS), and mixing to obtain a first mixed solution;

[0018] Mixing chitosan solution (CS) and silk fibroin (SF) to obtain a second mixed solution;

[0019] (S2) adding amino-modified basic copper phosphate nanoparticles (CP-NH2) to the first mixed solution prepared in step (S1), mixing well, adding chitosan solution, and post-treating to obtain a dense layer: basic copper phosphate nanoparticles / poly-L-glutamic acid / chitosan dense layer (as a barrier membrane);

[0020] (S3) adding amino-modified strontium-substituted hydroxyapatite (Sr-HA-NH2) to the second mixed solution prepared in step (S1), mixing well and pouring it onto the upper surface of the basic copper phosphate nanoparticles / poly-L-glutamic acid / chitosan dense layer prepared in step (S2), and post-treating to obtain a multifunctional composite guided bone regeneration membrane.

[0021] In one embodiment of the present invention, in step (S1), the ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, poly-L-glutamic acid and N-hydroxysuccinimide is 297.5:2 mL:36.5 mg;

[0022] The volume ratio of chitosan solution to silk fibroin was 1:1.

[0023] In one embodiment of the present invention, the preparation method of poly-L-glutamic acid comprises the following steps:

[0024] (S1011) tetrahydrofuran, triphosgene, and γ-benzyl-L-glutamic acid react under a nitrogen atmosphere and then post-treat to obtain a pretreated product;

[0025] (S1012) dissolving the pretreated product obtained in step (S1011) in tetrahydrofuran, adding n-hexane to dissolve, then recrystallizing at low temperature, and post-treating to obtain L-glutamic acid-N-carboxylic anhydride (BLG-NCA);

[0026] (S1013) mixing the L-glutamic acid-N-carboxylic anhydride prepared in step (S1012) with ethylene dichloride and triethylamine, and reacting the mixture, followed by post-treatment to obtain poly (L-glutamic acid benzyl ester);

[0027] Among them, triethylamine is used as an initiator to initiate the ring-opening polymerization reaction of L-glutamic acid-N-carboxylic anhydride;

[0028] (S1014) dissolving the poly (L-glutamic acid benzyl ester) prepared in step (S1013) in dichloroacetic acid, reacting with a HBr / Hac solution, and then post-treating to obtain poly (L-glutamic acid);

[0029] Among them, HBr / HAc removes the benzyl protecting group to obtain poly-L-glutamic acid.

[0030] In one embodiment of the present invention, the preparation method of silk fibroin comprises the following steps:

[0031] (S1021) placing the pretreated silkworm shells in a sodium carbonate solution, and performing post-processing after the reaction to obtain degummed silk;

[0032] (S1022) The degummed silk obtained in step (S1022) is placed in a lithium bromide solution, and subjected to post-treatment after the reaction to obtain silk fibroin.

[0033] In one embodiment of the present invention, in step (S2), the ratio of the amount of amino-modified basic copper phosphate nanoparticles, the first mixed solution and the chitosan solution is 3 mg: 1 mL: 1 mL;

[0034] The post-treatment is to volatilize naturally after molding, then immerse the mold in deionized water, and demould to obtain a dense layer of basic copper phosphate nanoparticles / poly-L-glutamic acid / chitosan.

[0035] In one embodiment of the present invention, the preparation method of amino-modified basic copper phosphate nanoparticles comprises the following steps:

[0036] (S2011) (NH4)2HPO4 solution was dropped into Cu(NO3)2·3H2O solution and the pH value was adjusted to 7. After the reaction, basic copper phosphate nanoparticles were obtained by post-treatment;

[0037] (S2012) dissolving 3-aminopropyltriethoxysilane in a mixed solvent of ethanol and water, then adding the basic copper phosphate nanoparticles prepared in step (S2011), and adjusting the pH to 10. After the reaction, post-treatment is performed to obtain amino-modified basic copper phosphate nanoparticles.

[0038] In one embodiment of the present invention, in step (S3), the ratio of the amount of amino-modified strontium-substituted hydroxyapatite to the second mixed solution is 2 mg:1 mL;

[0039] The post-treatment comprises freeze-drying at -20°C, immersing in deionized water, and demoulding to obtain a multifunctional composite guided bone regeneration membrane.

[0040] In one embodiment of the present invention, the preparation method of amino-modified strontium-substituted hydroxyapatite comprises the following steps:

[0041] (S2021) dissolving strontium hydroxide and calcium hydroxide and mixing them to obtain a suspension mixture;

[0042] (S2022) dropping a phosphoric acid solution into the suspension mixture prepared in step (S2021), adjusting the pH to 11, and performing post-treatment after the reaction to obtain strontium-substituted hydroxyapatite nanoparticles;

[0043] (S2023) dissolving 3-aminopropyltriethoxysilane in a mixed solvent of ethanol and water, then adding the strontium-substituted hydroxyapatite nanoparticles prepared in step (S2022), and adjusting the pH to 11. After the reaction, post-treatment is performed to obtain amino-modified strontium-substituted hydroxyapatite.

[0044] The third object of the present invention is to provide a multifunctional composite guided bone regeneration membrane for use in bone defect repair, for example, repair of alveolar bone defects.

[0045] The present invention proposes a multifunctional composite guided bone regeneration membrane. The present invention first grafts amino-modified copper phosphate (CP) with activated carboxyl groups on poly-L-glutamic acid (PLGA), and then chemically cross-links with chitosan (CS) solution, and prepares a CP / PLGA / CS dense layer with antibacterial and barrier effects by solvent casting. Amino-modified strontium-substituted hydroxyapatite (Sr-HA) is grafted with carboxyl groups on silk fibroin (SF), and then electrostatically interacts with -NH2 on CS, and a Sr-HA / SF / CS porous membrane is prepared by freeze-drying. The porous membrane biomimetic cytoplasmic matrix provides a physical space for the infiltration and growth of cells. Cu is doped in it. 2+ The guided bone regeneration membrane is endowed with antibacterial properties, Sr 2+ and Ca 2+ Both endowed the guided bone regeneration membrane with osteogenic properties.

[0046] In the present invention, a chemical cross-linked hydrogel membrane of synthetic poly L-glutamic acid (PLGA) and natural polysaccharide chitosan (CS) is used as a dense layer, and the activated carboxyl groups can cross-link amino-modified basic copper phosphate (CP) to solve the problem of Cu 2+ The sudden release problem is solved, aiming at antibacterial functional modification, continuous antibacterial effect, and at the same time improving the mechanical strength of the material to provide mechanical support and better repair bone defects; the natural polymer silk fibroin (SF) and chitosan (CS) are used to form a porous membrane through electrostatic action, aiming to simulate the cytoplasmic matrix of bone tissue and provide a microenvironment for the delivery of oxygen and nutrients; the rich free charges can adsorb amino-modified strontium to replace hydroxyapatite (Sr-HA), aiming to increase bone induction functional modification and better repair bone defects.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] (1) The porous layer of the present invention has good hydrophilicity and water absorption properties, and can provide sufficient space for cell growth.

[0049] (2) The present invention introduces the metal ion Cu 2+ , Cu 2+ It has antibacterial effects and is an essential cofactor for a variety of enzymes that are crucial for cell growth, differentiation, and survival in organisms such as bacteria, plants, and mammals.

[0050] (3) The present invention introduces metal ions Ca 2+ and Sr 2+ , Sr 2+ It can reduce bone resorption by promoting new tissue growth. 2+ It showed a dual beneficial effect, not only enhancing preosteoblast differentiation but also inhibiting osteoclast formation.

[0051] (4) The membrane material of the present invention has excellent biocompatibility and tissue affinity, can simulate the protein components in the extracellular matrix, promote cell growth and tissue repair, and has little rejection reaction caused by implantation into the organism. The degradation products are polysaccharides and amino acids, which are non-irritating to the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a flow chart for preparing a multifunctional composite guided bone regeneration membrane of the present invention;

[0053] Figure 2 This is a macroscopic photograph of the guided bone regeneration membrane;

[0054] Figure 3 Scanning electron images of guided bone regeneration membranes; A is a scanning electron image of the CP / PLGA / CS dense layer, and B is a scanning electron image of the Sr-HA / SF / CS porous layer;

[0055] Figure 4 Cu for guided bone regeneration membrane 2+ and Ca 2+ Release image; A is the Cu in the dense layer of CP / PLGA / CS 2+ Release image of Sr-HA / SF / CS porous layer. 2+ Release the image;

[0056] Figure 5 Images of the cell shielding effect of guided bone regeneration membranes; A shows the cell shielding effect of the dense layer of CP / PLGA / CS, and B shows the cell shielding effect of the porous layer of Sr-HA / SF / CS.

[0057] Figure 6 These are images of the antibacterial performance of guided bone regeneration membranes; A is the image of the antibacterial performance of the CP / PLGA / CS dense layer against Staphylococcus aureus, and B is the image of the antibacterial performance of the CP / PLGA / CS dense layer against Escherichia coli. DETAILED DESCRIPTION

[0058] The present invention provides a multifunctional composite guided bone regeneration membrane, which is composed of a dense layer and a porous layer; the porous layer is placed on the upper surface of the dense layer;

[0059] The dense layer is obtained by amidating chitosan and poly-L-glutamic acid and then doping amino-modified basic copper phosphate nanoparticles;

[0060] The porous layer is obtained by doping a mixture of silk fibroin and chitosan with amino-modified strontium-substituted hydroxyapatite.

[0061] The present invention provides a method for preparing a multifunctional composite guided bone regeneration membrane, comprising the following steps:

[0062] (S1) adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) to a poly-L-glutamic acid (PLGA) solution, and then adding N-hydroxysuccinimide (NHS), and mixing to obtain a first mixed solution;

[0063] Mixing chitosan solution (CS) and silk fibroin (SF) to obtain a second mixed solution;

[0064] (S2) adding amino-modified basic copper phosphate nanoparticles (CP-NH2) to the first mixed solution prepared in step (S1), mixing well, adding chitosan solution, and post-treating to obtain a dense layer: basic copper phosphate nanoparticles / poly-L-glutamic acid / chitosan dense layer;

[0065] (S3) adding amino-modified strontium-substituted hydroxyapatite (Sr-HA-NH2) to the second mixed solution prepared in step (S1), mixing well and pouring it onto the upper surface of the basic copper phosphate nanoparticles / poly-L-glutamic acid / chitosan dense layer prepared in step (S2), and post-treating to obtain a multifunctional composite guided bone regeneration membrane.

[0066] In one embodiment of the present invention, in step (S1), the ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, poly-L-glutamic acid and N-hydroxysuccinimide is 297.5:2 mL:36.5 mg;

[0067] The volume ratio of chitosan solution to silk fibroin was 1:1.

[0068] In one embodiment of the present invention, the preparation method of poly-L-glutamic acid comprises the following steps:

[0069] (S1011) tetrahydrofuran, triphosgene, and γ-benzyl-L-glutamic acid react under a nitrogen atmosphere and then post-treat to obtain a pretreated product;

[0070] (S1012) dissolving the pretreated product obtained in step (S1011) in tetrahydrofuran, adding n-hexane to dissolve, then recrystallizing at low temperature, and post-treating to obtain L-glutamic acid-N-carboxylic anhydride (BLG-NCA);

[0071] (S1013) mixing the L-glutamic acid-N-carboxylic anhydride prepared in step (S1012) with ethylene dichloride and triethylamine, and reacting the mixture, followed by post-treatment to obtain poly (L-glutamic acid benzyl ester);

[0072] Among them, triethylamine is used as an initiator to initiate the ring-opening polymerization reaction of L-glutamic acid-N-carboxylic anhydride;

[0073] (S1014) dissolving the poly (L-glutamic acid benzyl ester) prepared in step (S1013) in dichloroacetic acid, reacting with a HBr / Hac solution, and then post-treating to obtain poly (L-glutamic acid);

[0074] Among them, HBr / HAc removes the benzyl protecting group to obtain poly-L-glutamic acid.

[0075] In one embodiment of the present invention, the preparation method of silk fibroin comprises the following steps:

[0076] (S1021) placing the pretreated silkworm shells in a sodium carbonate solution, and performing post-processing after the reaction to obtain degummed silk;

[0077] (S1022) The degummed silk obtained in step (S1022) is placed in a lithium bromide solution, and subjected to post-treatment after the reaction to obtain silk fibroin.

[0078] In one embodiment of the present invention, in step (S2), the ratio of the amino-modified basic copper phosphate nanoparticles, the first mixed solution and the chitosan solution is 3 mg: 1 mL: 1 mL;

[0079] The post-treatment is to volatilize naturally after molding, then immerse the mold in deionized water, and demould to obtain a dense layer of basic copper phosphate nanoparticles / poly-L-glutamic acid / chitosan.

[0080] In one embodiment of the present invention, the preparation method of amino-modified basic copper phosphate nanoparticles comprises the following steps:

[0081] (S2011) (NH4)2HPO4 solution was dropped into Cu(NO3)2·3H2O solution and the pH value was adjusted to 7. After the reaction, basic copper phosphate nanoparticles were obtained by post-treatment;

[0082] (S2012) dissolving 3-aminopropyltriethoxysilane in a mixed solvent of ethanol and water, then adding the basic copper phosphate nanoparticles prepared in step (S2011), and adjusting the pH to 10. After the reaction, post-treatment is performed to obtain amino-modified basic copper phosphate nanoparticles.

[0083] In one embodiment of the present invention, in step (S3), the ratio of the amount of amino-modified strontium-substituted hydroxyapatite to the second mixed solution is 2 mg:1 mL;

[0084] The post-treatment comprises freeze-drying at -20°C, immersing in deionized water, and demoulding to obtain a multifunctional composite guided bone regeneration membrane.

[0085] In one embodiment of the present invention, the preparation method of amino-modified strontium-substituted hydroxyapatite comprises the following steps:

[0086] (S2021) dissolving strontium hydroxide and calcium hydroxide and mixing them to obtain a suspension mixture;

[0087] (S2022) dropping a phosphoric acid solution into the suspension mixture prepared in step (S2021), adjusting the pH to 11, and performing post-treatment after the reaction to obtain strontium-substituted hydroxyapatite nanoparticles;

[0088] (S2023) dissolving 3-aminopropyltriethoxysilane in a mixed solvent of ethanol and water, then adding the strontium-substituted hydroxyapatite nanoparticles prepared in step (S2022), and adjusting the pH to 11. After the reaction, post-treatment is performed to obtain amino-modified strontium-substituted hydroxyapatite.

[0089] The present invention provides an application of a multifunctional composite guided bone regeneration membrane in bone defect repair.

[0090] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0091] In the following examples, unless otherwise specified, all reagents used are commercially available reagents, and all detection means and methods used are conventional detection means and methods in the art.

[0092] Example 1

[0093] This embodiment provides a CP-NH2 nanoparticle and a preparation method thereof, which specifically includes the following steps:

[0094] (1) 4.84 g of copper nitrate (Cu(NO3)2·3H2O) and 1.32 g of diammonium hydrogen phosphate ((NH4)2HPO4) were dissolved in 100 ml of deionized water under magnetic stirring. The (NH4)2HPO4 solution was slowly dripped into the Cu(NO3)2·3H2O solution to produce a light blue precipitate immediately. The pH value of the system was adjusted to about 7 with ammonia water (25 wt%) and nitric acid solution (1 M). The mixed system was heated to 60 °C and maintained for 12 h under constant stirring. After the reaction product was allowed to stand for 2 h, the supernatant was poured off, the lower solid was filtered, and washed with deionized water and anhydrous ethanol, placed in a -20 °C refrigerator, and freeze-dried to obtain CP nanoparticles. 2.210 g of 3-aminopropyltriethoxysilane (APTES) was added to a mixed solvent of 500 mL of ethanol and water (v / v, 9:1) and stirred at room temperature for half an hour to obtain a mixed solution.

[0095] (2) 2.4 g of CP nanoparticles were added to the mixed solution prepared in step (1), and the pH value of the mixed system was adjusted to about 10 with ammonia water (25 wt%); after stirring at room temperature for 3 h, the reaction product was allowed to stand for 2 h, the supernatant was poured out, the lower solid was filtered and washed with anhydrous ethanol, and placed in a -20°C refrigerator for freezing. After freeze-drying, CP-NH2 nanoparticles were obtained, which were placed in a vacuum drying oven for storage (the reaction process is shown in formula (I)).

[0096]

[0097] Example 2

[0098] This embodiment provides a polyglutamic acid and a preparation method thereof, which specifically comprises the following steps:

[0099] (1) Add 500 ml of tetrahydrofuran to a flask, add 27 g of triphosgene and 45 g of γ-benzyl-L-glutamic acid in three portions under a N2 flow atmosphere, react at 50°C with stirring for about 30 min until the solution is clear, and continue to pass N2 to cool to room temperature; after the reaction is completed, pour the reaction solution into 2 L of ice-cold petroleum ether for sedimentation, dissolve the resulting sediment with 500 ml of ethyl acetate, transfer it to a 1000 ml separating funnel, wash it with deionized water, saturated NaHCO3 and saturated NaCl, respectively, transfer the supernatant to a conical flask, and dry it with anhydrous magnesium sulfate (MgSO4) to obtain a pretreated product;

[0100] (2) The pretreated product prepared in step (1) was dried at room temperature for 8 h, filtered using a G4 funnel, and the resulting solution was pumped dry with an oil pump. 60 ml of anhydrous tetrahydrofuran was added and dissolved at 40 ° C. 45 ml of anhydrous n-hexane was added. A small amount of solid precipitated. The temperature was raised to 60 ° C. and dissolved. After cooling to room temperature, it was placed in a -20 ° C refrigerator for recrystallization for 8 h, and then pumped dry with an oil pump to obtain L-glutamic acid-N-carboxylic anhydride (BLG-NCA).

[0101] (3) 20 g of the dried BLG-NCA prepared in step (2) was added to an ampoule with a magnetic pole, and 300 ml of dichloroethane and 24 ml of triethylamine solution were added in sequence. The mixture was stirred at room temperature for 6 h. After the reaction was completed, the reaction solution was precipitated with 3 L of ether. The obtained product was vacuum dried to obtain poly (benzyl L-glutamate) (PBLG).

[0102] (4) In a round-bottom flask, 10 g of the PBLG prepared in step (3) was dissolved in 100 ml of dichloroacetic acid, and 30 mL of HBr / Hac (33.wt%) solution was added. The reaction was carried out at 30° C. for 1.5 h. After the reaction was completed, the reaction solution was added to 1 L of ice-cold ether for precipitation. The resulting precipitate was dissolved in 15 mL of DMF and placed in a dialysis bag (MWCO7000). Dialysis was performed for three days, during which deionized water was replaced every 3 h. The dialyzate was freeze-dried to obtain polyglutamic acid (PLGA).

[0103] Example 3

[0104] This embodiment provides a Sr-HA-NH2 nanoparticle and a preparation method thereof, which specifically includes the following steps:

[0105] (1) 0.60 g of strontium hydroxide (Sr(OH)2) and 3.33 g of calcium hydroxide (Ca(OH)2) were dispersed in 100 ml of deionized water under magnetic stirring to obtain a suspension mixture;

[0106] (2) 3.30 g of phosphoric acid (H3PO4) was dissolved in 150 ml of deionized water under magnetic stirring; after stirring evenly, the mixture was slowly added dropwise to the suspension mixture prepared in step (1), and the pH value of the system was adjusted to about 11 with ammonia water (25 wt%) and nitric acid solution (1 M), and the mixture was heated to 100 ° C and maintained for 24 h under constant stirring; after the reaction product was allowed to stand for 2 h, the supernatant was poured off, the lower solid was centrifuged, washed with deionized water, placed in a -20 ° C refrigerator, and freeze-dried to obtain Sr-HA nanoparticles;

[0107] (3) 2.210 g of 3-aminopropyltriethoxysilane (APTES) was added to a mixed solvent of 500 mL of ethanol and water (v / v, 9:1) and stirred at room temperature for half an hour; then 5.25 g of Sr-HA nanoparticles were added, and the pH value of the mixed system was adjusted to about 11 with ammonia water (25 wt%) and stirred at room temperature for 3 h; the reaction product was allowed to stand for 2 days, the upper supernatant was poured out, the lower solid was centrifuged and washed with anhydrous ethanol, and placed in a -20 ° C refrigerator for freezing. After freeze-drying, Sr-HA-NH2 nanoparticles were obtained and stored in a vacuum drying oven (the reaction process is shown in formula (II)).

[0108]

[0109] Example 4

[0110] This embodiment provides a silk fibroin and a preparation method thereof, which specifically includes the following steps:

[0111] (1) Purification and preparation of silk fibroin: 10 g of chopped silkworm shells were added to a round-bottom flask equipped with a magnet, and 1 L of 0.02 M sodium carbonate solution was added. The mixture was stirred at 100 °C for 1 h. After cooling to room temperature, the degummed silk was washed with deionized water and dried in a 60 °C oven. The dried silk was then sealed and frozen in a -80 °C refrigerator for storage.

[0112] (2) Add 2 g of the degummed silk prepared in step (1) to a round-bottom flask equipped with a magnet, pour in 10 mL of 9 M LiBr solution, stir at 60 ° C for 4 h until the silk is completely dissolved, cool to room temperature, and place the reaction solution in a dialysis tape (MWCO 7000) for 2 days, changing the deionized water every 3 h. Filter the dialyzed solution for later use.

[0113] Example 5

[0114] This embodiment provides a multifunctional composite guided bone regeneration membrane and a preparation method thereof.

[0115] (1) Preparation of the dense layer, specifically comprising the following steps:

[0116] 1) Add 297.5 mg of EDC·HCl to 2 mL of 2% PLGA solution, stir at room temperature for 20 min, then add 36.5 mg of NHS and stir at room temperature for 8 h to obtain a primary mixed solution;

[0117] 2) CP-NH2 (prepared in Example 1) was added to the primary mixed solution prepared in step 1) at a concentration of 1.5 mg / mL, and dispersed evenly by ultrasonication for 30 seconds. The mixture was then quickly poured into 2 mL of a 2.5% CS solution and stirred to obtain a secondary mixed solution (CP / PLGA / CS suspension).

[0118] 3) pouring the secondary mixed solution prepared in step 2) into a 4 cm×4 cm polytetrafluoroethylene mold, and obtaining a CP / PLGA / CS dense layer after natural evaporation;

[0119] 4) Soaking the CP / PLGA / CS dense layer prepared in step 3) in deionized water for 2 days to remove small molecules such as EDC, NHS, and acetic acid, during which the deionized water was replaced every 4 hours. The layer was demolded to obtain a single-layer CP / PLGA / CS dense layer.

[0120] (2) Preparation of a multifunctional composite guided bone regeneration membrane, specifically comprising the following steps:

[0121] 1) Mixing equal volumes of a 2% SF solution (prepared in Example 4) and a 2% CS solution to obtain a first mixed solution;

[0122] 2) Sr-HA-NH2 (prepared in Example 3) was added to the first mixed solution prepared in step 1) at a concentration of 2 mg / mL and stirred for 4 h to obtain a second mixed solution (Sr-HA / SF / CS suspension);

[0123] 3) The second mixed solution prepared in step 2) was poured into a polytetrafluoroethylene mold containing the CP / PLGA / CS dense layer prepared in step (1), and placed in a -20°C refrigerator for freezing. After freeze-drying, the membrane was soaked in deionized water to remove acetic acid, and demolded to obtain a multifunctional composite guided bone regeneration membrane (CP / PLGA / CS dense layer and the Sr-HA / SF / CS porous layer on the upper surface of the CP / PLGA / CS dense layer).

[0124] The preparation process and mechanism diagram of this embodiment are as follows Figure 1 As shown, the macroscopic photograph of the prepared guided bone regeneration membrane is shown in Figure 2 As shown, the scanning electron image of the CP / PLGA / CS dense layer is shown in Figure 3 As shown in A, the scanning electron image of the Sr-HA / SF / CS porous layer is shown in Figure 3 As shown in B, the dense layer of CP / PLGA / CS Cu 2+ The release image is as follows Figure 4 As shown in A, the Sr-HA / SF / CS porous layer Ca 2 + Release image as Figure 4 As shown in B.

[0125] Furthermore, the CP / PLGA / CS dense layer and the Sr-HA / SF / CS porous layer were cut into 6 mm discs using a punch and placed in the cell chamber. 750 μL of MEM medium was added to the lower chamber, and 250 μL of 1×10 5 / mL L929 cell suspension was placed in a CO2 incubator and cultured at 37°C. On the 1st, 4th, and 7th day, the cell chamber was removed, the culture medium was removed, and the cells were washed twice with PBS solution. The cells were fixed with formaldehyde for 2 minutes at room temperature, and then the formaldehyde was removed and washed twice with PBS solution. 300μL DiO cell membrane stain was added, and the cells were incubated at 37°C in the dark for 10 minutes. The DiO stain was removed and washed twice with PBS solution. The migration of cells on the CP / PLGA / CS dense layer and the Sr-HA / SF / CS porous layer was then observed by confocal microscopy (as shown in Figure 2). Figure 5 shown).

[0126] In order to quantitatively evaluate the antibacterial properties of the CP / PLGA / CS dense layer, the antibacterial rate of the bacterial suspension was determined. 600 =0.1 (bacterial concentration is 1×10 8CFU / mL) of S. aureus and E. coli were cultured together with the C / PLGA / CS dense layer in a 96-well plate and incubated at 37°C for 48 h. During the first 12 h, the OD values ​​of the 96-well plate were measured every 2 h. 600 The absorbance value at the position was used to quantitatively analyze the bacterial concentration. 200 μL of bacterial solution was added to each well with a CP / PLGA / CS dense layer with a diameter of 6 mm, and a control group was used without the CP / PLGA / CS dense layer. Each group had 3 samples and the average value was taken (the results are shown in Figure 2). Figure 6 shown).

[0127] The present invention adopts the solution casting method to prepare the CP / PLGA / CS dense layer, pours the Sr-HA / SF / CS solution into the mold covered with the dense layer and freeze-dries it to obtain a multifunctional composite guided bone regeneration membrane; wherein, the dense layer is added with CP nanoparticles, which has antibacterial function and more effectively prevents the invasion of epithelial cells; the porous layer simulates the porous structure of the extracellular matrix, and Sr 2+ and Ca 2+ The invention can realize controlled release and also endow the porous layer with certain osteogenic function; the invention provides great application potential for the composite guided bone regeneration membrane in the field of bone defects.

[0128] A multifunctional composite guided bone regeneration membrane for repairing bone defects, the guided bone regeneration membrane consists of a dense layer and a porous layer. The dense layer is first constructed by solvent casting, and then the porous layer is constructed on the basis of the dense layer by freeze drying technology, finally obtaining a double-layer composite guided bone regeneration membrane. The multifunctional composite guided bone regeneration membrane prepared by the present invention has a dense layer blended with basic copper phosphate nanoparticles, which has antibacterial function and increases hydrophobicity to more effectively prevent the invasion of epithelial cells. The porous layer has a pore structure that simulates the extracellular matrix and can controllably release Sr 2+ and Ca 2+ Can accelerate the repair of bone defects.

[0129] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the explanations of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A multifunctional composite guided bone regeneration membrane, characterized in that: The multifunctional composite guided bone regeneration membrane consists of a dense layer and a porous layer; the porous layer is placed on the upper surface of the dense layer; The dense layer is obtained by amidating chitosan and poly-L-glutamic acid and then doping amino-modified basic copper phosphate nanoparticles; The porous layer is obtained by doping amino-modified strontium-substituted hydroxyapatite with a mixture of silk fibroin and chitosan; The multifunctional composite guided bone regeneration membrane was prepared by the following method: (S1) adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the poly-L-glutamic acid solution, then adding N-hydroxysuccinimide, and mixing to obtain a first mixed solution; Mixing the chitosan solution and the silk fibroin to obtain a second mixed solution; (S2) adding amino-modified basic copper phosphate nanoparticles to the first mixed solution prepared in step (S1), mixing well, adding chitosan solution, and post-treating to obtain a dense layer: basic copper phosphate nanoparticles / poly-L-glutamic acid / chitosan dense layer; (S3) adding the amino-modified strontium-substituted hydroxyapatite to the second mixed solution prepared in step (S1), mixing well, and then pouring it onto the upper surface of the basic copper phosphate nanoparticles / poly-L-glutamic acid / chitosan dense layer prepared in step (S2), and post-treating to obtain a multifunctional composite guided bone regeneration membrane; In step (S1), the ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, poly-L-glutamic acid, and N-hydroxysuccinimide is 297.5 mg:2 mL:36.5 mg; The volume ratio of chitosan solution to silk fibroin was 1:1; In step (S2), the ratio of amino-modified basic copper phosphate nanoparticles, the first mixed solution and the chitosan solution is 3 mg:1 mL:1 mL; The post-treatment is to volatilize naturally after molding, and then further immerse it in deionized water, and demould to obtain a dense layer of basic copper phosphate nanoparticles / poly-L-glutamic acid / chitosan; In step (S3), the ratio of amino-modified strontium-substituted hydroxyapatite to the second mixed solution is 2 mg:1 mL; The post-treatment comprises freeze-drying at -20°C, immersing in deionized water, and demoulding to obtain a multifunctional composite guided bone regeneration membrane.

2. The method for preparing a multifunctional composite guided bone regeneration membrane according to claim 1, characterized in that: The preparation method of poly-L-glutamic acid comprises the following steps: (S1011) Tetrahydrofuran, triphosgene, and γ-benzyl-L-glutamic acid react under a nitrogen atmosphere and then undergo post-treatment to obtain a pretreated product; (S1012) dissolving the pretreated product obtained in step (S1011) in tetrahydrofuran, adding n-hexane to dissolve, then recrystallizing at low temperature, and post-treating to obtain L-glutamic acid-N-carboxylic anhydride; (S1013) mixing the L-glutamic acid-N-carboxylic anhydride prepared in step (S1012) with ethylene dichloride and triethylamine, and reacting the mixture, followed by post-treatment to obtain poly (L-glutamic acid benzyl ester); (S1014) dissolving the poly (L-glutamic acid benzyl ester) prepared in step (S1013) in dichloroacetic acid, reacting with a HBr / Hac solution, and then post-treating to obtain poly (L-glutamic acid).

3. The method for preparing a multifunctional composite guided bone regeneration membrane according to claim 1, characterized in that: The preparation method of silk fibroin comprises the following steps: (S1021) placing the pretreated silkworm shells in a sodium carbonate solution, and performing post-processing after the reaction to obtain degummed silk; (S1022) placing the degummed silk obtained in step (S1021) in a lithium bromide solution, and performing post-treatment after the reaction to obtain silk fibroin.

4. The method for preparing a multifunctional composite guided bone regeneration membrane according to claim 1, characterized in that: The preparation method of amino-modified basic copper phosphate nanoparticles comprises the following steps: (S2011) (NH4)2HPO4 solution was dropped into Cu(NO3)2·3H2O solution and the pH value was adjusted to 7. After the reaction, basic copper phosphate nanoparticles were obtained by post-treatment; (S2012) dissolving 3-aminopropyltriethoxysilane in a mixed solvent of ethanol and water, then adding the basic copper phosphate nanoparticles prepared in step (S2011), and adjusting the pH to 10. After the reaction, post-treatment is performed to obtain amino-modified basic copper phosphate nanoparticles.

5. The method for preparing a multifunctional composite guided bone regeneration membrane according to claim 1, characterized in that: The preparation method of amino-modified strontium-substituted hydroxyapatite comprises the following steps: (S2021) dissolving strontium hydroxide and calcium hydroxide and mixing them to obtain a suspension mixture; (S2022) dropping a phosphoric acid solution into the suspension mixture prepared in step (S2021), adjusting the pH to 11, and performing post-treatment after the reaction to obtain strontium-substituted hydroxyapatite nanoparticles; (S2023) dissolving 3-aminopropyltriethoxysilane in a mixed solvent of ethanol and water, then adding the strontium-substituted hydroxyapatite nanoparticles prepared in step (S2022), and adjusting the pH to 11. After the reaction, post-treatment is performed to obtain amino-modified strontium-substituted hydroxyapatite.

6. Use of the multifunctional composite guided bone regeneration membrane according to claim 1 in the preparation of a drug for repairing bone defects.

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