WO2022116359A1 - 一种负载骨形成蛋白的水凝胶、制备方法及应用 - Google Patents

一种负载骨形成蛋白的水凝胶、制备方法及应用 Download PDF

Info

Publication number
WO2022116359A1
WO2022116359A1 PCT/CN2021/000150 CN2021000150W WO2022116359A1 WO 2022116359 A1 WO2022116359 A1 WO 2022116359A1 CN 2021000150 W CN2021000150 W CN 2021000150W WO 2022116359 A1 WO2022116359 A1 WO 2022116359A1
Authority
WO
WIPO (PCT)
Prior art keywords
bone
loaded
morphogenetic protein
hydrogel
bmp
Prior art date
Application number
PCT/CN2021/000150
Other languages
English (en)
French (fr)
Inventor
刘飞
张秀华
孙婷
凌沛学
陈勉
邵华荣
刘霞
刘英梅
郭新艳
何小华
Original Assignee
山东省药学科学院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 山东省药学科学院 filed Critical 山东省药学科学院
Publication of WO2022116359A1 publication Critical patent/WO2022116359A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
    • C08J3/246—Intercrosslinking of at least two polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/26—Mixtures of macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/02—Dextran; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2377/00—Characterised by the use of polyamides obtained by reactions forming a carboxylic amide link in the main chain; Derivatives of such polymers
    • C08J2377/04—Polyamides derived from alpha-amino carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2489/00—Characterised by the use of proteins; Derivatives thereof

Definitions

  • the invention belongs to the field of biological tissue engineering, and more particularly, relates to a hydrogel loaded with bone morphogenetic protein, a preparation method and an application.
  • Bone morphogenetic proteins are members of the TGF-beta superfamily, which are pleiotropic regulators of skeletal organogenesis and homeostasis. Among them, bone morphogenetic protein-2 (BMP-2) and bone morphogenetic protein-7 (BMP-7) have the best bone-inducing activity, which can induce bone marrow mesenchymal stem cells to differentiate into osteoblasts and increase the alkaline phosphatase of cells. Activity and levels of bone matrix mineralization that provide the primary signals for the differentiation of pluripotent cells into mineral-depositing osteoblasts. At present, BMP-2 has become the most commonly used protein bone graft substitute.
  • BMP is an unstable protein with a short half-life, maintaining its biological activity in the body for only a few minutes.
  • Continuous administration of large doses will not only increase the economic burden of patients, but also cause side effects such as heterotopic ossification, bone cysts, and inflammation. Therefore, in order to improve the patient's medication compliance, overcome the short half-life of BMP, and at the same time maintain a long-term stimulation effect at the defect site, and achieve effective bone healing, choosing an appropriate delivery system is the key to controlling the clinically required dose and release time.
  • hydrogels and BMP-2 are often used for compounding, but the reaction conditions are often not mild enough, or the hydrogels after cross-linking molding will have certain cytotoxicity, and there are problems such as poor biocompatibility or immunogenicity. .
  • the present invention provides a preparation method of a hydrogel loaded with bone morphogenetic protein, comprising:
  • the cross-linking agent, the water-soluble natural degradable polymer and the bone-forming protein are mixed and reacted in a liquid for injection or an aqueous buffer to obtain a bone-forming protein-loaded hydrogel.
  • the mixing reaction of the cross-linking agent, the water-soluble natural degradable polymer and the bone-forming protein in the liquid for injection includes:
  • the bone-forming protein is added to the cross-linking agent solution; the water-soluble natural degradable polymer is added to carry out the reaction.
  • the molecular weight of the water-soluble natural degradable polymer is 50-3 million Daltons, the content of the water-soluble natural degradable polymer is 50-200 mg/ml; the content of the cross-linking agent is 2 -10 ⁇ L/ml; the content of the bone morphogenetic protein is 0.5-5 mg/ml.
  • reaction temperature is 37-60°C, and the reaction time is 2-10h; preferably, the reaction temperature is 40-50°C, and the reaction time is 3-8h; further preferably, the reaction temperature is 45°C °C, the reaction time is 5h.
  • the cross-linking agent includes 1,4 butanediol diglycidyl ether, glutaraldehyde, N-hydroxysuccinimide ester, carbodiimide, genipin, cystamine, hexamethylene isocyanide
  • the water-soluble natural degradable polymer is one or more of polyglutamic acid, hyaluronic acid, xanthan gum, chitosan, pullulan, and carboxymethyl starch.
  • a bone morphogenetic protein-loaded hydrogel prepared by the above method. Further, the proliferation rate of cells incubated with the bone morphogenetic protein-loaded hydrogel is over 90%.
  • the hydrogel loaded with bone morphogenetic protein is implanted into mice, the alkaline phosphatase content in the blood of the mice is 80U/L-83U/L, and the muscle bag of the mice is New bone grows.
  • the bone morphogenetic protein release rates of the BMP-loaded hydrogel at 2d, 5d, 10d, 15d, 20d, 25d and 30d are 2-4%, 13-15%, 30-34%, respectively. , 48-51%, 63-67%, 70-73%, 76-80%.
  • the bone morphogenetic protein-loaded hydrogel prepared by the present invention has good biocompatibility and biodegradability, avoiding the need for a second operation to remove the implant material;
  • the bone morphogenetic protein-loaded hydrogel prepared by the present invention has good plasticity, can be directly injected into the bone defect site, completely fits the bone defect area, and has a small surgical wound;
  • the effective combination of water-soluble natural degradable polymer and bone morphogenetic protein does not affect the activity of the protein, can induce the growth of autologous cells at the site of bone injury, and promote the repair of bone tissue;
  • the degradation rate of the hydrogel can be controlled, thereby controlling the release rate of BMP, so that the BMP can be continuously released in the body, which solves the shortcomings of the short half-life of BMP and easy degradation in vivo;
  • the system is the adsorption between groups through electrostatic interaction, it is a stable release system, and there will be no sudden release or inability to release, which improves the utilization rate of bone morphogenetic protein and avoids the excessive concentration of bone morphogenetic protein. high incidence of complications;
  • the hydrogel forming time is obviously shortened compared with the hydrogel without BMP, and the hydrogel formed at the same time has a higher degree of cross-linking.
  • the method has the advantages of simple operation, mild preparation conditions, convenient and controllable, and continuous production.
  • Fig. 1 is the electron microscope picture of the polyglutamic acid hydrogel loaded with bone morphogenetic protein according to an embodiment of the present invention
  • FIG. 2 is an in vitro BMP release curve of a bone morphogenetic protein-loaded polyglutamic acid hydrogel according to an embodiment of the present invention
  • Fig. 3 is the cell proliferation ratio diagram of the polyglutamic acid hydrogel loaded with bone morphogenetic protein according to an embodiment of the present invention
  • Fig. 4 is the X-ray of the implantation site of the bone morphogenetic protein-loaded polyglutamic acid hydrogel according to an embodiment of the present invention after implantation in the muscle bag of mice;
  • Fig. 5 is a graph showing the content of alkaline phosphatase in serum after the bone morphogenetic protein-loaded polyglutamic acid hydrogel according to an embodiment of the present invention was implanted into the muscle bag of mice.
  • Fig. 6 is the HE staining diagram of the new bone after the bone morphogenetic protein-loaded polyglutamic acid hydrogel according to an embodiment of the present invention is implanted into the mouse muscle bag;
  • FIG. 7 is a Masson staining diagram of new bone after the bone morphogenetic protein-loaded polyglutamic acid hydrogel according to an embodiment of the present invention is implanted into a mouse muscle bag;
  • the present invention provides a method for preparing a bone-forming protein-loaded hydrogel, which comprises the following steps: mixing a cross-linking agent, a water-soluble natural degradable polymer and a bone-forming protein in a liquid for injection or an aqueous buffer to react to obtain a bone-loaded bone Protein hydrogels are formed.
  • the bone morphogenetic protein includes more than 30 related proteins, such as BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, etc.
  • the bone morphogenetic protein described in this application It is one or a mixture of two or more of the more than 30 related proteins.
  • the crosslinking agent is 1,4 butanediol diglycidyl ether, glutaraldehyde, N-hydroxysuccinimide ester, carbodiimide, genipin, cystamine, hexamethylene dicyanate, One or more of nitrogen diphenylphosphine, polyethylene glycol diacrylate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
  • the water-soluble natural degradable polymer is one or more of polyglutamic acid, hyaluronic acid, xanthan gum, chitosan, pullulan, and carboxymethyl starch.
  • the water-soluble natural degradable polymer is one or more of polyglutamic acid, hyaluronic acid, xanthan gum, chitosan, pullulan, and carboxymethyl starch.
  • the BMP-loaded hydrogel is directly formed by mixing the three raw materials, without the need to form a hydrogel first and then load the BMP.
  • the BMP-loaded hydrogel formed by the method of this example The loading rate was higher and the BMP loading was more uniform.
  • the molecular weight range of the water-soluble natural degradable polymer is 50-3 million Daltons, preferably 1.5-2.5 million Daltons.
  • the degree of cross-linking is extremely low, and it is easily degraded, the loss in the body is large, and the effect of sustained release during use cannot be achieved; when the molecular weight is greater than 3 million, the formation of The degree of cross-linking of the hydrogel is too large, it is relatively hard, does not flow, and is not easy to inject.
  • the mixing reaction of cross-linking agent, water-soluble natural degradable polymer and bone-forming protein in liquid for injection in the present application includes: dissolving the cross-linking agent in the water for injection or aqueous buffer to form a cross-linking agent solution; The bone-forming protein is added to the cross-linking agent solution; the water-soluble natural degradable polymer is added to carry out the reaction.
  • the method of this embodiment significantly shortens the formation time of the hydrogel system, and the formed hydrogel has a higher degree of cross-linking.
  • the water-soluble natural degradable polymer is negatively charged for the polyanion, and the bone morphogenetic protein is positively charged, and the two can act as polyelectrolytes, increasing the degree of crosslinking and shortening the gelation time.
  • the viscosity of the water-soluble natural degradable polymer itself makes the hydrogel adhere to the defect site and is not easy to run off.
  • This application can better induce the differentiation of mesenchymal stem cells into osteoblasts (rather than other cells), which matches the growth rate of new bone, and the new bone generated is in the number of collagen fibers, bone density, trabecular bone thickness, bone size The number of beams is closer to that of autologous bone.
  • the method is simple to operate, has high repeatability, can be continuously produced, and is suitable for large-scale production and sales.
  • the specific reaction steps are as follows: S100: the cross-linking agent is dissolved in the water for injection or aqueous buffer to form a cross-linking agent solution; S200: BMP is added to the cross-linking agent solution; S300: natural degradable The polymer is added to the solution formed in the step S200, and the reaction is performed.
  • BMP is added.
  • the natural degradable polymer is negatively charged as polyanion, and BMP is positively charged. Both of them can have polyelectrolyte effect, increase the degree of cross-linking, and shorten the gelation time. After adjusting the above reaction sequence, it was found that the viscosity of the solution would greatly increase, resulting in the inability to mix uniformly and affecting the reaction effect.
  • the content of the cross-linking agent is 2-10 ⁇ L/ml; in the S200, the content of the bone morphogenetic protein is 0.5-5 mg/ml;
  • the content of the natural degradable polymer is 50-200 mg/ml.
  • the degradation rate of the hydrogel can be controlled by adjusting the degree of cross-linking, that is, by adjusting the amount of cross-linking agent, reaction time and temperature, the degree of cross-linking can be controlled, thereby controlling the release rate of bone morphogenetic protein, so that bone morphogenetic protein can be released in vivo.
  • the sustained release solves the shortcomings of short half-life and easy degradation of bone morphogenetic protein in vivo, making it match the speed of new bone formation.
  • reaction conditions for the formation of the BMP-loaded hydrogel are as follows: the reaction temperature is 37-60 °C, and the reaction time is 2-10 h; preferably, the reaction temperature is 40-50 °C, and the reaction The time is 3-8h; further preferably, the reaction temperature is 45°C, and the reaction time is 4-6h.
  • the specific reaction time is related to the addition amount and temperature of the natural degradable polymer and cross-linking agent. release rate. Selecting this temperature range can keep the protein activity unchanged. If the temperature is too high or too low, the activity of the protein will be affected and the activity of the protein will be reduced.
  • the natural degradable polymer is sterilized before the reaction.
  • the specific sterilization method is as follows:
  • the sterilizing of the natural degradable polymer includes: irradiating the natural degradable polymer with ⁇ -rays, and the sterilizing dose is 4-10 kGy, preferably 6 kGy;
  • the bone-forming protein-loaded hydrogel will be used to treat diseases such as spinal fusion, there are strict requirements for bacterial content. Therefore, sterilization of the reactants before the reaction can better meet the follow-up requirements of the product. application to avoid situations that increase toxicity such as bacterial infection.
  • the present invention also provides a bone morphogenetic protein-loaded hydrogel prepared by the above method.
  • the bone morphogenetic protein-loaded hydrogel is cultured with cells, and the cell proliferation rate is over 90%.
  • the hydrogel loaded with bone morphogenetic protein is implanted into mice, the alkaline phosphatase content in the blood test serum of the mice is 80U/L-83U/L, and the muscle pockets of the mice have new bone grow out.
  • the release rates of the bone morphogenetic protein-loaded hydrogel at 2d, 5d, 10d, 15d, 20d, 25d and 30d were 2-4%, 13-15%, 30-34%, 48-48%, respectively. 51%, 63-67%, 70-73%, 76-80%.
  • the release rate refers to the percentage of the amount of BMP released to the total amount of BMP added, which is used to evaluate the sustained-release effect of BMP.
  • the degradation rate and BMP release rate of the BMP-loaded hydrogel of the present application are controllable, so that the BMP can be continuously released in the body, which matches the rate of new bone formation, and the new bone generated is in collagen fibers.
  • the number, bone mineral density, trabecular thickness, and number of trabecular bone were closer to those of autologous bone. If the release rate does not match (such as a sudden release in a short time), the old and new bones will not be well connected.
  • the application of the bone morphogenetic protein-loaded hydrogel prepared by the above method is used for the preparation of filling and repairing for the treatment of bone defect, nonunion, delayed bone union or nonunion, as well as spinal fusion,
  • the polyglutamate powder with a weight-average molecular weight of 1.5 million Daltons was sterilized by irradiation with ⁇ -rays, and the sterilization dose was 6 kGy.
  • the release rates of the 2d, 5d, 10d, 15d, 20d, 25d, and 30d of the products prepared by this method were 3.17%, 14.84%, 32.06%, 50.15%, 65.02%, 71.91%, and 78.73%, respectively;
  • the porosity of the product reached 96%;
  • the cell proliferation rate of 500 ⁇ g/mL of the product incubated with cells for 2 days was 111%; 4 weeks after the product was implanted in the muscle bag of mice, the small
  • the serum alkaline phosphatase content of the mice was 83 U/L, and X-ray films were performed. There was obvious new bone formation at the implantation site, and the bone shadow area of the new bone was 1.2cm*1.0cm.
  • the release rates of the 2d, 5d, 10d, 15d, 20d, 25d, and 30d of the products prepared by this method were 2.00%, 13.00%, 30.00%, 48.00%, 63.0%, 70.0%, and 76.0%, respectively;
  • the porosity of the product reached 97%;
  • the cell proliferation rate of the product incubated with cells at 500 ⁇ g/mL for 2 days was 108%; 4 weeks after the product was implanted in the muscle bag of mice, the small
  • the serum alkaline phosphatase content of the mice was 80U/L, and X-ray films were performed. There was obvious new bone formation at the implantation site, and the bone shadow area of the new bone was 1.1cm*1.0cm.
  • the release rates of bone morphogenetic protein on the 2d, 5d, 10d, 15d, 20d, 25d, and 30d of the products prepared by this method were 4.0%, 15.0%, 34.0%, 51.00%, 67.0%, 73.0%, and 80.0%, respectively;
  • the porosity of the product reached 95.6%;
  • the cell proliferation rate of the product incubated with cells at 500 ⁇ g/mL for 2 days was 109%; 4 weeks after the product was implanted into the muscle bag of mice, the cell proliferation rate was 109%.
  • the serum alkaline phosphatase content of the mice was 81 U/L, and X-ray films were performed. There was obvious new bone formation at the implantation site, and the bone shadow area of the new bone was 1.1cm*0.8cm.
  • the release rates of the 2d, 5d, 10d, 15d, 20d, 25d, and 30d of the products prepared by the method were 2.99%, 14.32%, 32.98%, 49.99%, 64.98%, 72.08%, and 77.98%, respectively;
  • the porosity of the product reached 97%;
  • the cell proliferation rate of the product incubated with cells at 500 ⁇ g/mL for 2 days was 112%; 4 weeks after the product was implanted into the muscle bag of mice, the small
  • the serum alkaline phosphatase content of the mice was 81 U/L, and X-ray films were performed. There was obvious new bone formation at the implantation site, and the bone shadow area of the new bone was 1.0cm*1.0cm.
  • the pullulan polysaccharide powder with a weight average molecular weight of 3 million Daltons is sterilized by irradiation with ⁇ -rays, and the sterilization dose is 8kGy;
  • the release rates of the 2d, 5d, 10d, 15d, 20d, 25d, and 30d of the products prepared by this method were 3.56%, 14.26%, 31.45%, 49.08%, 66.23%, 72.09%, and 79.22%, respectively;
  • the porosity of the product reached 97.2%;
  • the cell proliferation rate of the product incubated with cells at 500 ⁇ g/mL for 2 days was 106%; 4 weeks after the product was implanted in the muscle bag of mice, the small
  • the serum alkaline phosphatase content of the mice was 82 U/L, and X-ray films were performed. There was obvious new bone formation at the implantation site, and the bone shadow area of the new bone was 1.1cm*0.9cm.
  • BMP-2 protein 2.5 mg was added to the above polyglutamic acid hydrogel.
  • the release rates of the 2d, 5d, 10d, 15d, 20d, 25d, and 30d of the products prepared by this method were 30.53%, 66.55%, 77.32%, 83.22%, 86.99%, 87.92%, and 88.65%, respectively;
  • the porosity of the product reached 86.2%;
  • the cell proliferation rate of the product incubated with cells at 500 ⁇ g/mL for 2 days was 108%; 4 weeks after the product was implanted in mouse muscle bags, the small
  • the content of alkaline phosphatase in the serum of the mice was 60 U/L, and X-ray films were performed. There was obvious new bone formation at the implantation site, and the bone shadow area of the new bone was 0.5cm*0.5cm.
  • the weight-average molecular weight of the polyglutamate powder was 4 million Daltons, and the others were the same as in Example 1.
  • the release rates of the 2d, 5d, 10d, 15d, 20d, 25d, and 30d of the products prepared by this method were 10.53%, 26.55%, 27.32%, 28.22%, 28.99%, 29.92%, and 530.65%, respectively;
  • the porosity of the product reached 82%;
  • the cell proliferation rate of 500 ⁇ g/mL of the product incubated with cells for 2 days was 100%; after the product was implanted in mouse muscle bags for 4 weeks, the small
  • the serum alkaline phosphatase content of the mice was 56 U/L, and X-ray films were performed. There was obvious new bone formation at the implantation site, and the bone shadow area of the new bone was 0.4cm*0.5cm.
  • reaction conditions were that the reaction temperature was 80° C. and the reaction time was 3 h, and other conditions were the same as those in Example 1.
  • the release rates of the 2d, 5d, 10d, 15d, 20d, 25d, and 30d of the products prepared by this method were 0.53%, 8.09%, 18.22%, 25.33%, 30.09%, 35.92%, and 40.65%, respectively;
  • the porosity of the product reached 97.2%;
  • the cell proliferation rate of 500 ⁇ g/mL of the product incubated with cells for 2 days was 100%; 4 weeks after the product was implanted into the muscle bag of mice, the small The content of alkaline phosphatase in the serum of the mice was 42 U/L. X-ray films were performed, and there was no obvious new bone formation at the implantation site.
  • BMP was dissolved in water for injection to prepare a BMP solution with a concentration of 1 mg/mL, which was filtered and sterilized with a 0.22 ⁇ m filter.
  • the BMP-2 solution was directly injected into the muscle bag of the mice, and after 4 weeks, the serum alkaline phosphatase content of the mice was 67 U/L; X-ray films were performed, and new bone was formed at the implantation site, and new bone was formed.
  • the bone shadow area was 0.6cm*0.8cm, and there was inflammation at the implantation site.
  • Example 1 The BMP-loaded polyglutamic acid hydrogel obtained in Example 1 was characterized.
  • the prepared BMP-loaded polyglutamic acid hydrogel was photographed, and the hydrogel was milky white.
  • the prepared BMP-loaded polyglutamic acid hydrogel was pre-frozen at -80°C to a temperature slightly lower than the temperature required for freeze-drying, and then placed in a freeze-drying machine for freeze-drying treatment until drying was complete.
  • the surface and cross-sectional structure of the hydrogel were observed under a scanning electron microscope after the samples were sprayed with gold, as shown in Figure 1.
  • the porous network inside the gel enables the effective loading of bone-forming proteins.
  • the BMP-2-loaded hydrogel was placed in a dialysis bag, added with sterile PBS solution, and placed in a 37°C incubator.
  • the dialysate was taken at different time points and supplemented with an equal amount of PBS. After collecting the supernatant at all time points, the content of BMP-2 in the dialysate was detected by ELISA kit, and the cumulative release curve was calculated and drawn, as shown in Figure 2. It can be seen that the hydrogel loaded with BMP-2 has a stable release rate, and there will be no sudden release or failure to release, which can improve the utilization rate of BMP-2 and avoid complications caused by excessive BMP-2 concentration. .
  • the BMP-2-loaded hydrogel material was fully swollen/crushed by adding the medium. After centrifugation, the supernatant was taken to culture the cells, and the cell viability was measured at 24h and 48h. As shown in Figure 3, the BMP-2-loaded hydrogel can be seen. Has good cytocompatibility.
  • mice were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg), the outer left leg was sheared, routinely sterilized, and an incision of about 1 cm was made.
  • the experiment was divided into 3 groups, namely BMP-polyglutamic acid hydrogel group, polyglutamic acid hydrogel group, and sham operation group.
  • Corresponding materials were implanted according to the grouping situation, the muscles and skin were sutured in turn, and feeding was normal.
  • Antibiotics were injected intramuscularly for 3 consecutive days after surgery to prevent infection of the surgical area.
  • the implanted area was observed by ordinary X-ray film, as shown in Figure 4; blood was taken to measure the content of alkaline phosphatase in serum, as shown in Figure 5; the implantation site was dissected to observe the new bone formation, as shown in Figure 5.
  • Figure 6-7 through histological staining observation, HE staining showed that the quality of new bone was good, the number of trabecular bone was large, the thickness of trabecular bone was good, and there were a large number of active osteoblasts; Masson staining showed that the area of new collagen was large. It can be seen from the above characterization that the BMP-loaded hydrogel prepared by this method has the effects of low toxicity, high BMP loading rate, slow and stable release, and effective promotion of new bone formation.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Dermatology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Dispersion Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Materials For Medical Uses (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

本发明涉及属于生物组织工程领域，更具体地，涉及一种负载骨形成蛋白的水凝胶、制备方法及应用。所述方法包括：将交联剂、水溶性天然可降解聚合物以及骨形成蛋白于注射用液体或水性缓冲液中混合反应，得到负载骨形成蛋白的水凝胶。本发明制备的负载骨形成蛋白的水凝胶具有良好的生物相容性和生物降解性，避免了二次手术取出植入材。

Description

一种负载骨形成蛋白的水凝胶、制备方法及应用 技术领域
本发明涉及属于生物组织工程领域，更具体地，涉及一种负载骨形成蛋白的水凝胶、制备方法及应用。
背景技术
目前，由创伤、感染、肿瘤或先天性遗传疾病引起的骨损伤是一种常见的骨科疾病。骨组织本身具有一定的自动修复和再生的能力，但是，大面积的骨缺损需要通过外部干预的方法进行治疗，其再生修复仍然是临床医学面临的一项重大挑战。目前，自体骨移植和异体骨移植是治疗骨损伤的常用方法。作为金标准的自体骨移植具有骨引导、骨诱导和骨再生的三大特性，但自体骨的获取数量受到很大限制，同时对患者来说存在创伤大、手术时间长和风险大等问题。对于异体骨移植来说，不可预知的骨整合效果和潜在的对于致死性病原体的传播是限制其广泛应用的最大瓶颈。
骨形成蛋白(BMP)是TGF-β超级家族成员，它们是骨骼器官形成发生和保持稳态的多效调节剂。其中尤以骨形成蛋白-2(BMP-2)、骨形成蛋白-7(BMP-7)诱骨活性最好，能够诱导骨髓间充质干细胞向成骨细胞分化，提高细胞的碱性磷酸酶活性和骨基质矿化水平，为多能细胞分化成矿物质沉积成骨细胞提供主要信号。目前BMP-2己成为最常用的蛋白质植骨替代物，经美国国家药物监督管理局(FDA)以及欧洲药品管理局(EMA)准许用于医治脊柱融合，也是目前唯一能避免骨移植的治疗方案。但BMP是一种不稳定的蛋白质，半衰期短，在体内维持其生物活性的时间仅数分钟。若大剂量持续给药，不仅增加患者经济负担，也会引起异位骨化、骨囊肿、炎症等副反应。因此为了提高患者用药顺应性，克服BMP半衰期短等问题，同时使其在缺损部位保持长期刺激作用，实现有效骨愈合，选择合适的递送系统是控制临床要求剂量和释放时间的关键。现有技术多利用水凝胶与BMP-2复合，但是其反应条件往往不够温和，或者交联成型后的水凝胶会带有一定的细胞毒性，存在生物相容性差或免疫原性等问题。
发明内容
为了解决上述技术问题，本发明提供了一种负载骨形成蛋白的水凝胶的制备方法，包括：
将交联剂、水溶性天然可降解聚合物以及骨形成蛋白于注射用液体或水性缓冲液中混合反应，得到负载骨形成蛋白的水凝胶。
进一步地，所述将交联剂、水溶性天然可降解聚合物以及骨形成蛋白于注射用液体中混合反应包括：
将所述交联剂溶于所述注射用水或水性缓冲液中形成交联剂溶液；
将所述骨形成蛋白加入所述交联剂溶液；加入所述水溶性天然可降解聚合物，进行反应。
进一步地，所述水溶性天然可降解聚合物的分子量为50-300万道尔顿，所述水溶性天然可降解聚合物的含量为50-200mg/ml；所述交联剂的含量为2-10μL/ml；所述骨形成蛋白的含量为0.5-5mg/ml。
进一步地，所述反应温度为37-60℃，反应时间为2-10h；优选的，所述反应温度为40-50℃，反应时间为3-8h；进一步优选的，所述反应温度为45℃，反应时间为5h。
进一步地，所述交联剂包括为1，4丁二醇二缩水甘油醚、戊二醛、N-羟基琥珀酰亚胺酯、碳化二亚胺、京尼平、胱胺、己异二氰酸酯、叠氮二苯基膦、聚乙二醇二丙烯酸酯、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐中的一种或多种；所述水溶性天然可降解聚合物为聚谷氨酸、透明质酸、黄原胶、壳聚糖、普鲁兰多糖、羧甲基淀粉中的一种或多种。
另一方面，还提供一种负载骨形成蛋白的水凝胶，由上述的方法制备。进一步地，经所述负载骨形成蛋白的水凝胶培育细胞增殖率为90％以上。
进一步地，将所述负载骨形成蛋白的水凝胶植入小鼠，所述小鼠的血测血清中碱性磷酸酶含量为80U/L-83U/L，且所述小鼠肌袋处长出新骨。
进一步地，所述负载骨形成蛋白的水凝胶在2d、5d、10d、15d、20d、25d及30d的骨形成蛋白的释放率分别为2-4％、13-15％、30-34％、48-51％、63-67％、70-73％、76-80％。
本发明具有以下优点：
1、本发明制备的负载骨形成蛋白的水凝胶具有良好的生物相容性和生物降解性，避免了二次手术取出植入材料；
2、本发明制备的负载骨形成蛋白的水凝胶可塑性好，可直接注射到骨缺损部位，与骨缺损区域完全契合，手术创口小；
3、水溶性天然可降解聚合物与骨形成蛋白的有效结合不影响蛋白活性，可诱导骨损伤部位自体细胞生长，促进骨组织的修复；
4、通过调节交联度可控制水凝胶的降解速度，从而控制BMP的释放速度，使BMP在体内持续释放，解决了BMP体内半衰期较短易降解等缺点；
5、由于该体系是基团间通过静电作用的吸附，是稳定的释放体系，不会发生突释或无法释放的情况，提高了骨形成蛋白的利用率，同时也避免了骨形成蛋白浓度过高引发的并发症；
6、加入骨形成蛋白后，水凝胶成型时间与不加骨形成蛋白的水凝胶成型时间相比明显缩短，同时形成的水凝胶交联度更高。
7、该方法操作简单，制备条件温和，方便可控，能连续生产。
本发明的其它特征和优点将在随后的说明书中阐述，并且，部分地从说明书中变得显而易见，或者通过实施本发明而了解。本发明的目的和其他优点可通过在说明书、权利要求书以及附图中所特别指出的结构来实现和获得。
附图说明
附图用来提供对本发明技术方案的进一步理解，并且构成说明书的一部分，与本申请的实施例一起用于解释本发明的技术方案，并不构成对本发明技术方案的限制。
图1是本发明一个实施例的负载骨形成蛋白的聚谷氨酸水凝胶的电镜图；
图2是本发明一个实施例的负载骨形成蛋白的聚谷氨酸水凝胶的体外BMP释放曲线；
图3是本发明一个实施例的负载骨形成蛋白的聚谷氨酸水凝胶的细胞增值比例图；
图4是本发明一个实施例的负载骨形成蛋白的聚谷氨酸水凝胶植入小鼠肌袋后植入部位的X光；
图5是本发明一个实施例的负载骨形成蛋白的聚谷氨酸水凝胶植入小鼠肌袋后血清中碱性磷酸酶含量图。
图6是本发明一个实施例的负载骨形成蛋白的聚谷氨酸水凝胶植入小鼠肌袋后新生骨的HE染色图；
图7是本发明一个实施例的负载骨形成蛋白的聚谷氨酸水凝胶植入小鼠肌袋后新生骨的Masson染色图；
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例的附图，对本发明实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明的 一部分实施例，而不是全部的实施例。基于所描述的本发明的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明提供一种负载骨形成蛋白的水凝胶的制备方法，包括：将交联剂、水溶性天然可降解聚合物以及骨形成蛋白于注射用液体或水性缓冲液中混合反应，得到负载骨形成蛋白的水凝胶。
该骨形成蛋白包括30多种相关蛋白，如BMP-1、BMP-2、BMP-3、BMP-4、BMP-5、BMP-6、BMP-7等等，本申请所述的骨形成蛋白为该30多种相关蛋白中的一种或两种以上混合物。
所述交联剂为1，4丁二醇二缩水甘油醚、戊二醛、N-羟基琥珀酰亚胺酯、碳化二亚胺、京尼平、胱胺、己异二氰酸酯、叠氮二苯基膦、聚乙二醇二丙烯酸酯、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐中的一种或多种。
所述水溶性天然可降解聚合物为聚谷氨酸、透明质酸、黄原胶、壳聚糖、普鲁兰多糖、羧甲基淀粉中的一种或多种。
所述水溶性天然可降解聚合物为聚谷氨酸、透明质酸、黄原胶、壳聚糖、普鲁兰多糖、羧甲基淀粉中的一种或多种。
本实施例通过将三种原料混合直接形成负载骨形成蛋白的水凝胶，无需先形成水凝胶，再对骨形成蛋白进行负载，采用本实施例方法形成的负载骨形成蛋白的水凝胶负载率更高，且骨形成蛋白负载更加均匀。
所述水溶性天然可降解聚合物的分子量范围50-300万道尔顿，优选为150-250万道尔顿。
当水溶性天然可降解聚合物的分子量小于50万道尔顿时，交联度极低，且极易降解，体内流失量大，使用过程无法达到缓释的效果；当分子量大于300万时，形成的水凝胶交联度太大，比较硬，不流动，不易注射。
本申请将交联剂、水溶性天然可降解聚合物以及骨形成蛋白于注射用液体中混合反应包括：将所述交联剂溶于所述注射用水或水性缓冲液中形成交联剂溶液；将所述骨形成蛋白加入所述交联剂溶液；加入所述水溶性天然可降解聚合物，进行反应。相比先形成水凝胶，再负载骨形成蛋白的方法，采用本实施例的方法明显缩短该水凝胶体系的形成时间，且形成的水凝胶交联度更高。这主要是由于水溶性天然可降解聚合物为聚阴离子带负电荷，骨形成蛋白带正电荷，两者可发生聚电解质作用，增加交联度，缩短成胶时间。同时，水溶性天然可降解聚合物本身的粘性，使水凝胶黏附于缺损部位，不易流失。本申请可以更好的 诱导间充质干细胞向成骨细胞分化(而非其他细胞)，与新骨生长速率相匹配，生成的新骨在胶原纤维数、骨密度、骨小梁厚度、骨小梁数量等方面跟自体骨更接近。
该方法操作简单，且重复性高，可连续生产，适合规模化生产及销售等。具体地反应步骤如下：S100：所述交联剂溶于所述注射用水或水性缓冲液中形成交联剂溶液；S200：将骨形成蛋白加入所述交联剂溶液；S300：将天然可降解聚合物加入所述步骤S200形成的溶液中，进行反应即可。
在形成水凝胶之前，加入骨形成蛋白，天然可降解聚合物为聚阴离子带负电荷，骨形成蛋白带正电荷，两者可发生聚电解质作用，增加交联度，缩短成胶时间。对上述反应顺序进行调整后发现，溶液粘度会大大增加，导致无法混合均匀，影响反应效果。
进一步地，所述S100中，所述交联剂含量为2-10μL/ml；所述S200中，所述骨形成蛋白的含量为0.5-5mg/ml；
所述S300中，所述天然可降解聚合物含量为50-200mg/ml。
通过调节交联度可控制水凝胶的降解速度，即通过对交联剂的用量、反应时间及温度进行调节，控制交联度，从而控制骨形成蛋白的释放速度，使骨形成蛋白在体内持续释放，解决了骨形成蛋白体内半衰期较短易降解等缺点，使其与新骨生成的速度匹配。
进一步地，所述负载骨形成蛋白的水凝胶形成的反应条件如下：所述反应温度为37-60℃，反应时间为2-10h；优选的，所述反应温度为40-50℃，反应时间为3-8h；进一步优选的，所述反应温度为45℃，反应时间为4-6h。具体的反应时间与天然可降解聚合物和交联剂的添加量及温度有关，添加量越多，温度越高，交联时间越长，交联度则越大，越不易降解，从而控制BMP的释放率。选择该温度范围内可保持蛋白活性不变，温度过高或过低，都会影响蛋白的活性，使蛋白活性降低。
本发明的另一实施例，在反应之前，对所述天然可降解聚合物进行灭菌。具体的灭菌方法如下：
所述对天然可降解聚合物进行灭菌包括：利用γ-射线进行辐照所述天然可降解聚合物，所述灭菌剂量为4-10kGy，优选为6kGy；
由于该负载骨形成蛋白的水凝胶将被用于医治脊柱融合等疾病，对于细菌含量等具有严格的要求，因此，反应前对反应物进行灭菌处理，可以更好的满足该产品的后续应用，避免发生细菌感染等增加毒性的情况。
本发明还提供一种负载骨形成蛋白的水凝胶，由上述方法制备。所述负载骨形成蛋白的水凝胶与细胞进行培育，细胞的增殖率为90％以上。
可见，该产品对于细胞非常友好，毒性较低。将所述负载骨形成蛋白的水凝胶植入小鼠，所述小鼠的血测血清中碱性磷酸酶含量为80U/L-83U/L，且所述小鼠肌袋处有新骨长出。
所述负载骨形成蛋白的水凝胶在2d、5d、10d、15d、20d、25d及30d的骨形成蛋白的释放率分别为2-4％、13-15％、30-34％、48-51％、63-67％、70-73％、76-80％。
其中释放率指的是BMP释放出的量占加入BMP总量的百分比，用来评价BMP的缓释效果。
也就是说，本申请的负载骨形成蛋白的水凝胶的降解速度和BMP释放速度可控，使骨形成蛋白在体内持续释放，其与新骨生成的速度匹配，生成的新骨在胶原纤维数、骨密度、骨小梁厚度、骨小梁数量等方面跟自体骨更接近。如果释放率不匹配(如短时间突然释放)，将会导致新旧骨无法良好衔接。本发明的另一实施例中，由上述方法制备的负载骨形成蛋白的水凝胶的应用，用于制备治疗骨缺损、骨不连、骨延迟愈合或不愈合的填充修复，以及脊柱融合、关节融合及矫形植骨修复，颌面部的骨缺损修复，牙周组织骨缺损、口腔种植、唇腭裂、牙槽嵴裂骨质缺失修复中一种或多种的药物。
为了对本发明有更加清楚的理解，现详细说明具体实施例。
实施例1
(1)原料的灭菌
取重均分子量为150万道尔顿的聚谷氨酸盐粉末用γ-射线进行辐照灭菌，灭菌剂量6kGy。
(2)水凝胶制备以下过程在无菌条件下进行。
将1，4丁二醇二缩水甘油醚0.01mL，溶于5mL重蒸馏水，加入2.5mgBMP-2蛋白。称取上述灭菌后的重均分子量为150万道尔顿的聚谷氨酸盐粉末0.25g，加入上述溶液中，搅拌均匀后静止片刻至气泡消失，37℃保温10h。
该方法制备得到的产品第2d、5d、10d、15d、20d、25d、30d骨形成蛋白的释放率分别为3.17％、14.84％、32.06％、50.15％、65.02％、71.91％、78.73％；该产品的孔隙率(孔隙所占面积/总面积)达到96％；500μg/mL该产品与细胞孵育2天的细胞增殖率为111％；该产品植入小鼠肌袋4周后，所述小鼠的血清中碱性磷酸酶含量为83U/L，进行X射线拍片，植入部位有明显新骨形成，且新骨的骨阴影面积为1.2cm*1.0cm。
实施例2
(1)原料的灭菌
取重均分子量为250万道尔顿的透明质酸用γ-射线进行辐照灭菌，灭菌剂量10kGy；
(2)水凝胶制备以下过程在无菌条件下进行。
将戊二醛0.05mL，溶于5mL硼酸盐缓冲液中，加入BMP-2 25mg。称取上述灭菌后的重均分子量为250万道尔顿的透明质酸1.0g，加入上述溶液中，搅拌均匀后静止片刻至气泡消失，60℃保温2h。该方法制备得到的产品第2d、5d、10d、15d、20d、25d、30d骨形成蛋白的释放率分别为2.00％、13.00％、30.00％、48.00％、63.0％、70.0％、76.0％；该产品的孔隙率(孔隙所占面积/总面积)达到97％；500μg/mL该产品与细胞孵育2天的细胞增殖率为108％；该产品植入小鼠肌袋4周后，所述小鼠的血清中碱性磷酸酶含量为80U/L，进行X射线拍片，植入部位有明显新骨形成，且新骨的骨阴影面积为1.1cm*1.0cm。
实施例3
(1)原料的灭菌
取重均分子量为200万道尔顿的聚谷氨酸用γ-射线进行辐照灭菌，灭菌剂量4kGy；
(2)水凝胶制备以下过程在无菌条件下进行。
将1，4丁二醇二缩水甘油醚0.02mL，溶于5mL重蒸馏水中，加入10mgBMP-3。称取上述灭菌后的重均分子量为200万道尔顿的聚谷氨酸0.5g，加入上述溶液中，搅拌均匀后静止片刻至气泡消失，40℃保温8h。该方法制备得到的产品第2d、5d、10d、15d、20d、25d、30d骨形成蛋白的释放率分别为4.0％、15.0％、34.0％、51.00％、67.0％、73.0％、80.0％；该产品的孔隙率(孔隙所占面积/总面积)达到95.6％；500μg/mL该产品与细胞孵育2天的细胞增殖率为109％；该产品植入小鼠肌袋4周后，所述小鼠的血清中碱性磷酸酶含量为81U/L，进行X射线拍片，植入部位有明显新骨形成，且新骨的骨阴影面积为1.1cm*0.8cm。
实施例4
(1)原料的灭菌
取重均分子量为50万道尔顿的壳聚糖用γ-射线进行辐照灭菌，灭菌剂量5kGy；
(2)水凝胶制备以下过程在无菌条件下进行。
将1，4丁二醇二缩水甘油醚0.03mL，溶于5mL水性缓冲液中，加入15mgBMP-5溶液。称取上述灭菌后的重均分子量为50万道尔顿的壳聚糖0.6g，加入上述溶液中，搅拌均匀后静止片刻至气泡消失，50℃保温3h。该方法制备得到的产品第2d、5d、10d、15d、20d、25d、30d骨形成蛋白的释放率分别为2.99％、14.32％、32.98％、49.99％、64.98％、 72.08％、77.98％；该产品的孔隙率(孔隙所占面积/总面积)达到97％；500μg/mL该产品与细胞孵育2天的细胞增殖率为112％；该产品植入小鼠肌袋4周后，所述小鼠的血清中碱性磷酸酶含量为81U/L，进行X射线拍片，植入部位有明显新骨形成，且新骨的骨阴影面积为1.0cm*1.0cm。
实施例5
(1)原料的灭菌
取重均分子量为300万道尔顿的普鲁兰多糖粉末用γ-射线进行辐照灭菌，灭菌剂量8kGy；
(2)水凝胶制备以下过程在无菌条件下进行。
将1，4丁二醇二缩水甘油醚0.04mL，溶于5mL重蒸馏水中，加入4mg BMP-7溶液。称取上述灭菌后的重均分子量为300万道尔顿的普鲁兰多糖0.8g，加入上述溶液中，搅拌均匀后静止片刻至气泡消失，45℃保温5h。该方法制备得到的产品第2d、5d、10d、15d、20d、25d、30d骨形成蛋白的释放率分别为3.56％、14.26％、31.45％、49.08％、66.23％、72.09％、79.22％；该产品的孔隙率(孔隙所占面积/总面积)达到97.2％；500μg/mL该产品与细胞孵育2天的细胞增殖率为106％；该产品植入小鼠肌袋4周后，所述小鼠的血清中碱性磷酸酶含量为82U/L，进行X射线拍片，植入部位有明显新骨形成，且新骨的骨阴影面积为1.1cm*0.9cm。
对比例1
(1)原料灭菌与实施例1相同；
(2)聚谷氨酸水凝胶制备
将1，4丁二醇二缩水甘油醚0.01mL，溶于重蒸馏水，称取上述灭菌后的重均分子量为150万道尔顿的聚谷氨酸盐粉末0.25g，加入上述溶液中，搅拌均匀后静止片刻至气泡消失，37℃保温15h形成聚谷氨酸水凝胶。
(3)骨形成蛋白的吸附
将2.5mg BMP-2蛋白加入上述聚谷氨酸水凝胶中。该方法制备得到的产品第2d、5d、10d、15d、20d、25d、30d骨形成蛋白的释放率分别为30.53％、66.55％、77.32％、83.22％、86.99％、87.92％、88.65％；该产品的孔隙率(孔隙所占面积/总面积)达到86.2％；500μg/mL该产品与细胞孵育2天的细胞增殖率为108％；该产品植入小鼠肌袋4周后，所述小鼠的血清中碱性磷酸酶含量为60U/L，进行X射线拍片，植入部位有明显新骨形成，且新骨的骨阴影面积为0.5cm*0.5cm。
对比例2
聚谷氨酸盐粉末的重均分子量为400万道尔顿，其他与实施例1相同。
该方法制备得到的产品第2d、5d、10d、15d、20d、25d、30d骨形成蛋白的释放率分别为10.53％、26.55％、27.32％、28.22％、28.99％、29.92％、530.65％；该产品的孔隙率(孔隙所占面积/总面积)达到82％；500μg/mL该产品与细胞孵育2天的细胞增殖率为100％；该产品植入小鼠肌袋4周后，所述小鼠的血清中碱性磷酸酶含量为56U/L，进行X射线拍片，植入部位有明显新骨形成，且新骨的骨阴影面积为0.4cm*0.5cm。
对比例3
反应条件为反应温度80℃，反应时间3h，其他与实施例1相同。
该方法制备得到的产品第2d、5d、10d、15d、20d、25d、30d骨形成蛋白的释放率分别为0.53％、8.09％、18.22％、25.33％、30.09％、35.92％、40.65％；该产品的孔隙率(孔隙所占面积/总面积)达到97.2％；500μg/mL该产品与细胞孵育2天的细胞增殖率为100％；该产品植入小鼠肌袋4周后，所述小鼠的血清中碱性磷酸酶含量为42U/L，进行X射线拍片，植入部位无明显新骨形成。
对比例4
BMP用注射用水溶解，制成浓度为1mg/mL的BMP溶液，0.22μm滤头过滤除菌后。将BMP-2溶液直接注射于小鼠肌袋，4周后，所述小鼠的血清中碱性磷酸酶含量为67U/L；进行X射线拍片，植入部位有新骨形成，且新骨的骨阴影面积为0.6cm*0.8cm，且植入部位存在炎症。
对实施例1得到的负载骨形成蛋白的聚谷氨酸水凝胶进行表征。
(1)成品图
将制备的负载骨形成蛋白的聚谷氨酸水凝胶进行拍照，该水凝胶呈乳白色。
(2)扫描电镜
将所制备的负载骨形成蛋白的聚谷氨酸水凝胶在-80℃里预冻至温度稍低于冷冻干燥所需温度后，放入冷冻干燥机进行冷冻干燥处理，至干燥完全。将样品经过喷金后在扫描电子显微镜下观察水凝胶的表面和截面结构，如图1所示。该凝胶内部的多孔网络，实现对骨形成蛋白的有效负载。
(3)体外释放：30天释放率
将负载BMP-2的水凝胶置于透析袋中，加入无菌PBS溶液，置于37℃培养箱中，分别在不同时间点取透析液中，并补足等量的PBS。待收集完全部时间点的上清液后，用 ELISA试剂盒分别检测透析液中BMP-2的含量，计算并绘制累计释放曲线，如图2所示。可见，负载BMP-2的水凝胶具有稳定的释放率，不会发生突释或无法释放的情况，可提高BMP-2的利用率，同时也可避免BMP-2浓度过高引发的并发症。
(4)细胞毒性实验
将负载BMP-2的水凝胶材料加培养基充分溶胀/打碎，离心后取上清培养细胞，测24h、48h细胞存活率，如图3所示，可见负载BMP-2的水凝胶具有良好的细胞相容性。
(5)异位成骨活性
小鼠经戊巴比妥钠(40mg/kg)腹腔注射麻醉，后左腿外侧剪毛，常规消毒，作约1cm的切口，切开皮肤后分离外侧肌肉间隙陷窝。实验分3组，分别为BMP-聚谷氨酸水凝胶组、聚谷氨酸水凝胶组、假手术组。按分组情况分别植入相应的材料，依次缝合肌肉和皮肤，正常喂养。术后连续3天肌肉注射抗生素，以防止术区感染。
术后4周对植入区域进行普通X射线拍片观察，如图4所示；取血测血清中碱性磷酸酶含量，如图5所示；解剖植入部位，观察新骨形成情况，如图6-7所示，通过组织学染色观察，HE染色说明新生骨质量好，骨小梁数量多，骨小梁厚度好，有大量活跃的成骨细胞；Masson染色说明新生胶原面积大。通过上述表征可知，该方法制备的负载骨形成蛋白的水凝胶具有毒性低、骨形成蛋白负载率高、释放缓慢稳定以及有效促进新骨形成的效果。
虽然本发明所揭露的实施方式如上，但所述的内容仅为便于理解本发明而采用的实施方式，并非用以限定本发明。任何本发明所属领域内的技术人员，在不脱离本发明所揭露的精神和范围的前提下，可以在实施的形式及细节上进行任何的修改与变化，但本发明的专利保护范围，仍须以所附的权利要求书所界定的范围为准。

Claims (10)

  1. 一种负载骨形成蛋白的水凝胶的制备方法，其特征在于，包括：将交联剂、水溶性天然可降解聚合物以及骨形成蛋白于注射用液体或水性缓冲液中混合反应，得到负载骨形成蛋白的水凝胶。
  2. 根据权利要求1所述的负载骨形成蛋白的水凝胶的制备方法，其特征在于，所述将交联剂、水溶性天然可降解聚合物以及骨形成蛋白于注射用液体中混合反应包括：
    将所述交联剂溶于所述注射用水或水性缓冲液中形成交联剂溶液；将所述骨形成蛋白加入所述交联剂溶液；加入所述水溶性天然可降解聚合物，进行反应。
  3. 根据权利1所述的负载骨形成蛋白的水凝胶的制备方法，其特征在于，所述水溶性天然可降解聚合物的分子量为50-300万道尔顿，所述水溶性天然可降解聚合物含量为50-200mg/ml；所述交联剂的含量为2-10μL/ml；所述骨形成蛋白的含量为0.5-5mg/ml。
  4. 根据权利要求1所述的负载骨形成蛋白的水凝胶的制备方法，其特征在于，所述反应温度为37-60℃，反应时间为2-10h；优选的，所述反应温度为40-50℃，反应时间为3-8h；进一步优选的，所述反应温度为45℃，反应时间为5h。
  5. 根据权利要求1-4任一项所述的负载骨形成蛋白的水凝胶的制备方法，其特征在于，所述交联剂为1，4丁二醇二缩水甘油醚、戊二醛、N-羟基琥珀酰亚胺酯、碳化二亚胺、京尼平、胱胺、己异二氰酸酯、叠氮二苯基膦、聚乙二醇二丙烯酸酯、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐中的一种或多种；所述水溶性天然可降解聚合物为聚谷氨酸、透明质酸、黄原胶、壳聚糖、普鲁兰多糖、羧甲基淀粉中的一种或多种。
  6. 一种负载骨形成蛋白的水凝胶，其特征在于，由权利要求1-5任一项权利要求所述的方法制备。
  7. 根据权利要求6所述的负载骨形成蛋白的水凝胶，其特征在于，经所述负载骨形成蛋白的水凝胶培育的细胞增殖率为90％以上。
  8. 根据权利要求6所述的负载骨形成蛋白的水凝胶，其特征在于，将所述负载骨形成蛋白的水凝胶植入小鼠，所述小鼠的血测血清中碱性磷酸酶含量为80U/L-83 U/L，且所述小鼠肌袋处长出新骨。
  9. 根据权利要求6所述的负载骨形成蛋白的水凝胶，其特征在于，所述负载骨形成蛋白的水凝胶在2d、5d、10d、15d、20d、25d及30d的骨形成蛋白的释放率分别为2-4％、13-15％、30-34％、48-51％、63-67％、70-73％及76-80％。
  10. 一种由权利要求1-5任一项所述方法制备的负载骨形成蛋白的水凝胶的应用，其特征在于，用于制备治疗骨缺损、骨不连、骨延迟愈合或不愈合的填充修复，以及脊柱融合、关节融合及矫形植骨修复，颌面部的骨缺损修复，牙周组织骨缺损、口腔种植、唇腭裂、牙槽嵴裂骨质缺失修复中一种或多种的药物。
PCT/CN2021/000150 2020-12-02 2021-07-13 一种负载骨形成蛋白的水凝胶、制备方法及应用 WO2022116359A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011387383.4 2020-12-02
CN202011387383.4A CN112521638A (zh) 2020-12-02 2020-12-02 一种负载骨形成蛋白的水凝胶、制备方法及应用

Publications (1)

Publication Number Publication Date
WO2022116359A1 true WO2022116359A1 (zh) 2022-06-09

Family

ID=74996039

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/000150 WO2022116359A1 (zh) 2020-12-02 2021-07-13 一种负载骨形成蛋白的水凝胶、制备方法及应用

Country Status (2)

Country Link
CN (1) CN112521638A (zh)
WO (1) WO2022116359A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115845148A (zh) * 2022-12-07 2023-03-28 中国医学科学院北京协和医院 一种温敏可注射壳聚糖/丝素蛋白/水滑石复合水凝胶及其制备方法和应用
CN116903884A (zh) * 2023-06-06 2023-10-20 山东丰金美业科技有限公司 一种透明质酸-聚谷氨酸水凝胶及其制备方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112521638A (zh) * 2020-12-02 2021-03-19 山东省药学科学院 一种负载骨形成蛋白的水凝胶、制备方法及应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101036807A (zh) * 2007-04-24 2007-09-19 浙江大学 注射型藻酸钙/骨形态发生蛋白复合材料及制备方法
CN105837682A (zh) * 2015-11-25 2016-08-10 天津中津生物发展有限公司 一种bmp-3蛋白及其作为骨修复注射剂的制备方法及应用
US10016534B2 (en) * 2008-11-17 2018-07-10 Gel-Del Technologies, Inc. Protein biomaterial and biocoacervate vessel graft systems and methods of making and using thereof
EP3003026B1 (en) * 2013-03-14 2019-07-17 Tricol Biomedical, Inc. Biocompatible and bioabsorbable derivatized chitosan compositions
CN111658820A (zh) * 2020-05-07 2020-09-15 广州创赛生物医用材料有限公司 一种促进骨再生的可注射水凝胶及其制备方法
CN112521638A (zh) * 2020-12-02 2021-03-19 山东省药学科学院 一种负载骨形成蛋白的水凝胶、制备方法及应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012162840A1 (en) * 2011-06-03 2012-12-06 Frank Gu Polysaccharide-based hydrogel polymer and uses thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101036807A (zh) * 2007-04-24 2007-09-19 浙江大学 注射型藻酸钙/骨形态发生蛋白复合材料及制备方法
US10016534B2 (en) * 2008-11-17 2018-07-10 Gel-Del Technologies, Inc. Protein biomaterial and biocoacervate vessel graft systems and methods of making and using thereof
EP3003026B1 (en) * 2013-03-14 2019-07-17 Tricol Biomedical, Inc. Biocompatible and bioabsorbable derivatized chitosan compositions
CN105837682A (zh) * 2015-11-25 2016-08-10 天津中津生物发展有限公司 一种bmp-3蛋白及其作为骨修复注射剂的制备方法及应用
CN111658820A (zh) * 2020-05-07 2020-09-15 广州创赛生物医用材料有限公司 一种促进骨再生的可注射水凝胶及其制备方法
CN112521638A (zh) * 2020-12-02 2021-03-19 山东省药学科学院 一种负载骨形成蛋白的水凝胶、制备方法及应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115845148A (zh) * 2022-12-07 2023-03-28 中国医学科学院北京协和医院 一种温敏可注射壳聚糖/丝素蛋白/水滑石复合水凝胶及其制备方法和应用
CN116903884A (zh) * 2023-06-06 2023-10-20 山东丰金美业科技有限公司 一种透明质酸-聚谷氨酸水凝胶及其制备方法

Also Published As

Publication number Publication date
CN112521638A (zh) 2021-03-19

Similar Documents

Publication Publication Date Title
Bai et al. Bioactive hydrogels for bone regeneration
WO2022116359A1 (zh) 一种负载骨形成蛋白的水凝胶、制备方法及应用
Tao et al. Applications of chitin and chitosan nanofibers in bone regenerative engineering
Saravanan et al. Chitosan based biocomposite scaffolds for bone tissue engineering
Zhang et al. Stem cell-friendly scaffold biomaterials: applications for bone tissue engineering and regenerative medicine
US10442182B2 (en) In vivo live 3D printing of regenerative bone healing scaffolds for rapid fracture healing
Deng et al. Advanced applications of cellulose-based composites in fighting bone diseases
US7727542B2 (en) Bioactive materials, methods of making bioactive materials and method of use thereof
D’este et al. Hydrogels in calcium phosphate moldable and injectable bone substitutes: Sticky excipients or advanced 3-D carriers?
Albu et al. Collagen-based drug delivery systems for tissue engineering
US20090017093A1 (en) Gelatinous material for filling bone and/or cartilage defects
US20070248675A1 (en) Composite comprising polysaccharide-functionalized nanoparticle and hydrogel matrix, a drug delivery system and a bone defect replacement matrix for sustained release comprising the same, and the preparation method thereof
US20230069580A1 (en) Chemically cross-linked hydrogel and its microspheres, preparation method and application
Tong et al. Synthesis of and in vitro and in vivo evaluation of a novel TGF-β1-SF-CS three-dimensional scaffold for bone tissue engineering
Li et al. Biomimetic methacrylated gelatin hydrogel loaded with bone marrow mesenchymal stem cells for bone tissue regeneration
Zhang et al. Application of chitosan with different molecular weights in cartilage tissue engineering
Li et al. Injectable thermosensitive chitosan/gelatin-based hydrogel carried erythropoietin to effectively enhance maxillary sinus floor augmentation in vivo
Yan et al. Controlled release of BMP-2 from a heparin-conjugated strontium-substituted nanohydroxyapatite/silk fibroin scaffold for bone regeneration
KR20080017161A (ko) 다당류-기능화 나노입자 및 수화젤 담체를 포함하는복합체, 이를 포함하는 서방형 약물전달 제제, 뼈충진제 및이들의 제조방법
US20100143439A1 (en) Hybrid Biomimetic Particles, Methods of Making Same and Uses Therefor
Lee et al. Highly elastic 3D-printed gelatin/HA/placental-extract scaffolds for bone tissue engineering
Jagga et al. Chitosan-based scaffolds in tissue engineering and regenerative medicine
CN108770341A (zh) 具有钙涂层的骨空隙填充物
Kesharwani et al. Tissue regeneration properties of hydrogels derived from biological macromolecules: A review
US20090291113A1 (en) Osteogenic composition comprising a growth factor, a soluble cation salt and organic support

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21899403

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21899403

Country of ref document: EP

Kind code of ref document: A1