WO2023169074A1 - 一种骨粘接剂、其制备方法及应用 - Google Patents

一种骨粘接剂、其制备方法及应用 Download PDF

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WO2023169074A1
WO2023169074A1 PCT/CN2023/070599 CN2023070599W WO2023169074A1 WO 2023169074 A1 WO2023169074 A1 WO 2023169074A1 CN 2023070599 W CN2023070599 W CN 2023070599W WO 2023169074 A1 WO2023169074 A1 WO 2023169074A1
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bone
preparation
hydrogel
zif
polyvinyl alcohol
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PCT/CN2023/070599
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English (en)
French (fr)
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陈陶
胡杉杉
王珊
翟启明
季平
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重庆医科大学
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    • 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
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • 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
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block 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
    • 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

Definitions

  • the present disclosure relates to the technical field of medical materials, and specifically to a bone adhesive, its preparation method and application.
  • Fractures are one of the most common trauma injuries, endangering the health of tens of millions of people around the world every year. Among them, severe high fractures or comminuted fractures will significantly increase the difficulty of surgery and increase the incidence of delayed healing and poor bone healing. In addition, large bone defects caused by trauma or tumors are also a long-term clinical challenge.
  • bone cement/bone glue as an adjunct to adhesion and splicing of complex fractures may be an attractive approach with the potential to change surgical strategies for the treatment of highly comminuted fractures.
  • PMMA polymethylmethacrylate
  • CPC calcium phosphate
  • CA cyanoacrylate
  • the present disclosure provides a method for preparing a bone adhesive, which includes: using polyvinyl alcohol and levodopa to react to obtain an L-DP polymer, and reacting the L-DP polymer with ZIF-8, so that the L-DP polymer is The phenolic hydroxyl group chelates with zinc ions.
  • the mass ratio of the L-DP polymer to ZIF-8 is 100:1-2.
  • the L-DP polymer is obtained by reacting polyvinyl alcohol with a molecular weight of 85-124 kDa and levodopa.
  • the preparation method of the L-DP polymer includes: after dissolving polyvinyl alcohol, reacting it with levodopa in the presence of a catalyst, and preparing the reacted solution to form a gel.
  • the preparation process of the gel includes: dialyzing the reacted solution for 2-3 days, and then performing rotary evaporation and freeze-drying.
  • reaction temperature of polyvinyl alcohol and levodopa is 75-85°C, and the reaction time is 15-20 h.
  • reaction temperature of polyvinyl alcohol and levodopa is 78-82°C, and the reaction time is 12-18 hours.
  • the polyvinyl alcohol is dissolved in an organic solvent under heating conditions, and the organic solvent is dimethyl sulfoxide.
  • a protective gas is introduced during the reaction.
  • the catalyst used is sodium hydrogen sulfate hydrate.
  • the mass ratio of sodium bisulfate hydrate to polyvinyl alcohol is 3-4:1.
  • the aqueous solution formed by re-dissolving the gel is reacted with the aqueous solution formed by ZIF-8, and the reaction time is 0.5-1 h.
  • a solution obtained by reacting the gel with ZIF-8 is prepared to form a hydrogel.
  • the gel is sterilized by gamma ray irradiation and the raw materials are further cross-linked to form a gel.
  • the concentration of the aqueous solution formed by the gel is 150-250 mg/mL, and the concentration of the aqueous solution formed by ZIF-8 is 25-100 mg/mL.
  • the present disclosure also provides a bone adhesive prepared by the above preparation method.
  • the bone adhesive is in the form of a hydrogel.
  • the present disclosure also provides the use of the above-mentioned bone adhesive in preparing bone tissue repair materials.
  • the present disclosure also provides a biomedical material prepared by the above preparation method.
  • the present disclosure also provides the use of the bone adhesive or the biomedical material for bone tissue bonding, bone tissue repair and/or bone tissue healing.
  • the present disclosure also provides a method for treating bone trauma-related diseases, including:
  • the bone adhesive or the biomedical material is administered to a subject in need thereof.
  • the disease related to bone trauma includes at least one of fracture or bone defect.
  • Figure 1 is a scanning electron microscope image of ZIF-8 nanoparticles
  • Figure 2 shows the porous surface morphology formed under the electron microscope of L-DP gel
  • Figure 3 shows the porous surface morphology of L-DPZ hydrogel under an electron microscope
  • Figure 4 is the elemental analysis diagram of L-DPZ hydrogel
  • FIG. 5 shows the Fourier transform infrared spectrum test (FT-IR) of L-DP and L-DPZ hydrogels
  • Figure 6 shows the X-ray photoelectron spectroscopy (XPS) images of L-DP and L-DPZ hydrogels
  • Figure 7 is a diagram showing the self-healing behavior of two pieces of L-DPZ hydrogel of different colors after being cut off and combined;
  • Figure 8 shows the results of the strain amplitude scanning test (1-1000%) of L-DPZ hydrogel at a fixed angular frequency (10rad/s) and 37°C;
  • Figure 9 shows the results of the continuous cyclic strain test (5% strain ⁇ 600% strain ⁇ 5% strain) of L-DPZ hydrogel at a constant angular frequency (10 rad/s);
  • Figure 10 shows the shear thinning test results of L-DPZ hydrogel
  • Figure 11 shows the use of dumbbells to test the bonding strength of L-DPZ hydrogel to bovine bone slices
  • Figure 12 shows the bonding strength of L-DPZ hydrogel to bovine bone slices tested by humans
  • Figure 13 is a diagram showing the bonding effect of L-DPZ hydrogel on the truncated bovine tibia
  • Figure 14 is a bone chip shear force test diagram, where a is a schematic diagram of the bone chip shear force test method, and b is a representation of the bone chip shear force test data);
  • Figure 15 shows the bone block tensile force test diagram, in which a is a schematic diagram of the bone block tensile force test method, and b is the bone block tensile force test data representation);
  • Figure 16 is a test chart of the adhesion ability of PVA, L-DP and L-DPZ2 hydrogels to polytetrafluoroethylene and glass, where a and b are respectively PVA, L-DP and L-DPZ2 hydrogels to polytetrafluoroethylene and glass.
  • c and d are a schematic diagram of the testing method and data characterization of the bonding ability of PVA, L-DP and L-DPZ2 hydrogels on glass;
  • Figure 17 is a test chart of the bonding ability of L-DPZ hydrogel to bovine tooth fragments
  • Figure 18 shows the ability of L-DPZ hydrogel to bond and reduce dislocated pig teeth
  • Figure 19 shows the bonding performance test chart after mixing L-DPZ hydrogel and bone powder
  • Figure 20 shows the compressive strength and three-point bending strength test diagrams of L-DP and L-DPZ hydrogel and bone powder mixtures
  • Figure 21 shows the in vitro degradation rate detection results of L-DP and L-DPZ hydrogels
  • Figure 22 shows the in vivo degradation rate detection results of L-DP and L-DPZ hydrogels
  • Figure 23 shows the CCK8 activity test results of rBMSC
  • Figure 24 shows the ALP staining and ARS staining of the osteogenic performance of rBMSCs by L-DPZ hydrogel
  • Figure 25 is a qPCR test result of the osteogenic performance of L-DPZ hydrogel on rBMSC
  • Figure 26 is a comparison chart of the osteogenic performance test results of different proportions of L-DPZ hydrogel
  • Figure 27 is an operation diagram for testing the osteogenic ability of L-DPZ hydrogel in animals
  • Figure 28 is a micro-CT comparison of the bone formation effects in New Zealand rabbits after 4 and 8 weeks in the blank group, pure bone meal group, L-DP hydrogel and bone meal mixed group, and L-DPZ hydrogel and bone meal mixed group;
  • Figure 29 is a VG staining comparison of the osteogenic effects in New Zealand rabbits after 4 and 8 weeks in the blank group, pure bone meal group, L-DP hydrogel and bone meal mixed group, and L-DPZ hydrogel and bone meal mixed group.
  • One embodiment of the present disclosure provides a method for preparing a bone adhesive, which includes the following steps:
  • the reaction between polyvinyl alcohol and levodopa is used to obtain L-DP polymer, and the hydrogen bonds on polyvinyl alcohol are used to introduce levodopa into the long chain of polyvinyl alcohol, so that levodopa is grafted on polyvinyl alcohol to obtain L-DP polymer.
  • DP polymer The reaction between polyvinyl alcohol and levodopa is used to obtain L-DP polymer, and the hydrogen bonds on polyvinyl alcohol are used to introduce levodopa into the long chain of polyvinyl alcohol, so that levodopa is grafted on polyvinyl alcohol to obtain L-DP polymer.
  • levodopa L-3,4-dihydroxy-phenylalanine (L-DOPA)
  • L-DOPA levodopa, L-3,4-dihydroxy-phenylalanine
  • the preparation method of L-DP polymer includes: after dissolving polyvinyl alcohol, reacting with levodopa in the presence of a catalyst, and preparing the reacted solution to form a gel for subsequent introduction of ZIF- 8.
  • L-DP polymer is produced using a molecular weight of 85-124kDa (for example, 90-120kDa, 95-115kDa or 100-110kDa, such as 85kDa, 90kDa, 95kDa, 100kDa, 105kDa, 110kDa, 115kDa, 120kDa, or any two endpoint values)
  • the numerical interval between is obtained by reacting polyvinyl alcohol and levodopa, and the molar ratio of polyvinyl alcohol and levodopa is controlled to 4-7:1, such as 4:1, 5:1, 6:1, 7: 1, etc., or the numerical interval between any two endpoint values.
  • the polyvinyl alcohol is dissolved in an organic solvent under heating conditions.
  • the organic solvent is dimethyl sulfoxide (DMSO).
  • the catalyst used is sodium hydrogen sulfate hydrate.
  • the amount of catalyst used is not limited.
  • the mass ratio of sodium bisulfate hydrate to polyvinyl alcohol can be controlled to 3-4:1, such as 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5 :1, 3.6:1, 3.7:1, 3.8:1, 3.9:1 or 4.0:1, or the numerical range between any two endpoint values.
  • the reaction temperature of polyvinyl alcohol and levodopa is 75-85°C, and the reaction time is 15-20 h.
  • the reaction temperature of polyvinyl alcohol and levodopa is 78-82°C, and the reaction time is 12-18 hours.
  • the reaction temperature of polyvinyl alcohol and levodopa may be 77-83°C, 79-82°C or 80-81°C, such as 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82 °C, 83°C, 84°C or 85°C, etc., or the numerical interval between any two endpoint values, or any value between the above adjacent temperature values; the reaction time can be, for example, 14-19h, 15-18h Or 16-17h, such as 12h, 13h, 14h, 15h, 16h, 17h, 18h, etc., or a numerical interval between any two endpoint values, or any value between the above adjacent time values.
  • a protective gas is introduced during the reaction to prevent L-DOPA from being oxidized during the reaction.
  • the type of protective gas is not limited and can be nitrogen or other inert gases.
  • the gel preparation process includes: dialyzing the reacted solution for 2-3 days, and then performing rotary evaporation and freeze-drying.
  • rotary evaporation can be replaced by other evaporation forms that can remove most of the water; freeze-drying can also be replaced by other dry forms.
  • the L-DP polymer is reacted with ZIF-8, and zinc ions in ZIF-8 are used to form metal phenolic coordination bonds with levodopa to introduce zinc ions.
  • ZIF-8 uses zinc ions as a linker to sustainably release Zn 2+ and can play a positive role in osteogenesis, angiogenesis and antibacterial processes.
  • the aqueous solution formed by re-dissolving the gel is reacted with the aqueous solution formed by ZIF-8, and the reaction time is 0.5-1h; the solution after the reaction between the gel and ZIF-8 is prepared to form a hydrogel.
  • the gel with ZIF-8 use gamma ray irradiation (15Gmy) for 8 hours to sterilize and further cross-link the two to form a gel.
  • the final product L-DPZ may not exist in the form of a hydrogel.
  • the mass ratio of the control gel and ZIF-8 is 100:1-2, such as 100:1.1, 100:1.3, 100:1.4, 100:1.5, 100:1.6, 100:1.7, 100:1.8, 100:1.9 or 100:2.0, or an interval value between any two of the above endpoints.
  • the concentration of the aqueous solution formed by the gel is 150-250 mg/mL, such as 160-240 mg/mL, 170-230 mg/mL or 180-220 mg/mL, such as 150 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190mg/mL, 200mg/mL, 210mg/mL, 220mg/mL, 230mg/mL, 240mg/mL or 250mg/mL, or an interval value between any two of the above endpoints.
  • the aqueous solution formed by ZIF-8 has a concentration of 25-100 mg/mL, such as 30-90 mg/mL, 40-80 mg/mL, or 50-70 mg/mL, such as 26 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL , 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL or 100mg/mL, or the interval value between any two of the above endpoints.
  • the introduction amount of ZIF-8 the product can have excellent bone bonding properties while ensuring that the toxicity of the product meets the requirements. If the introduction amount of ZIF-8 is too large, it will lead to excessive toxicity; if ZIF- If the introduction amount of 8 is too small, the bone bonding performance of the product will be significantly reduced.
  • One embodiment of the present disclosure also provides a bone adhesive, which can be prepared by the above preparation method. Since this bone adhesive has excellent bone bonding properties and good biocompatibility, it can be used in the preparation of bone tissue repair materials to promote bone bonding and bone repair of severe bone trauma such as complex fractures and bone defects. healing process and has broad clinical application prospects.
  • the mass ratio of L-DP polymer formed from polyvinyl alcohol and levodopa to ZIF-8 is 100:1-2.
  • One embodiment of the present disclosure provides a biomedical material prepared by the above preparation method.
  • One embodiment of the present disclosure also provides a bone adhesive or biomedical material for use in bone tissue bonding, bone tissue repair, and/or bone tissue healing.
  • One embodiment of the present disclosure also provides a method for treating bone trauma-related diseases, including:
  • the bone trauma-related disease includes at least one of a fracture or a bone defect.
  • the present disclosure provides a bone adhesive and a preparation method thereof, which improves the bone adhesiveness, biocompatibility and osteogenic properties of the adhesive.
  • This disclosure grafts levodopa on polyvinyl alcohol and uses the phenolic hydroxyl group on levodopa to combine with ZIF-8 to form a multifunctional bionic ZIF-8 bone glue. It uses metal-organic framework ZIF-8 as the core to integrate The wet adhesive properties of a large number of catechol-structured levodopa (L-DOPA) molecules are used as a carrier, using the biocompatible polymer polyvinyl alcohol (PVA) as a carrier to construct a bio-adhesion and biophase material with good bone adhesion.
  • L-DOPA catechol-structured levodopa
  • PVA biocompatible polymer polyvinyl alcohol
  • L-DPZ bone glue A new type of bone glue (L-DPZ bone glue) that integrates capacitance, osteogenic properties, injectability and other functions, can promote the bone bonding and bone healing process of severe bone trauma such as complex fractures and bone defects, and has a wide range of clinical applications Application prospects.
  • This embodiment provides a method for preparing a bone adhesive, which includes the following steps:
  • step (1) Weigh the lyophilized hydrogel in step (1), dissolve it in deionized water at a concentration of 200 mg/mL, and add ZIF-8 at the same time to make the L-DP polymer:ZIF8 mass ratio reach 100:1; stir with Stir the mixture thoroughly and react for 1 hour. After the reaction, use gamma ray irradiation (15Gmy) for 8 hours to sterilize and further cross-link the two to form a gel, represented by L-DPZ1.
  • gamma ray irradiation 15Gmy
  • Example 2 The only difference from Example 1 is that the mass ratio of L-DP polymer:ZIF8 reaches 100:2, and the product is represented by L-DPZ2.
  • This embodiment provides a method for preparing a bone adhesive, which includes the following steps:
  • step (1) Weigh the freeze-dried hydrogel in step (1), dissolve it in deionized water at a concentration of 150 mg/mL, and add ZIF-8 at the same time to make the mass ratio of L-DP:ZIF8 reach 100:1. Use a stirrer to completely Stir evenly and react for 0.5h. After the reaction, use gamma ray irradiation (15Gmy) for 8h to sterilize and further cross-link the two to form a gel.
  • gamma ray irradiation 15Gmy
  • This embodiment provides a method for preparing a bone adhesive, which includes the following steps:
  • step (1) Weigh the freeze-dried hydrogel in step (1), dissolve it in deionized water at a concentration of 250 mg/mL, and add ZIF-8 at the same time to make the mass ratio of L-DP:ZIF8 reach 100:2. Use a stirrer to completely Stir evenly and react for 1 hour. After the reaction, use gamma ray irradiation (15Gmy) for 8 hours to sterilize and further cross-link the two to form a gel.
  • gamma ray irradiation 15Gmy
  • the adhesive of commercially available medical adhesive ⁇ -cyanoacrylate (Guangzhou Baiyun Medical Adhesive Co., Ltd., medical anastomotic type) was mixed with the L-DPZ1 hydrogel from Example 1 and the L-DPZ1 hydrogel from Example 2.
  • the adhesive properties of DPZ2 hydrogel were compared.
  • Example 1 The only difference from Example 1 is that the mass ratio of L-DP:ZIF8 is 100:4, represented by L-DPZ3 hydrogel.
  • ZIF8 is uniform in size, basically between 80-100nm, with no particles that are too large or too small.
  • both L-DP and L-DPZ gels show porous morphology, and the uniformity of the materials is good.
  • FIG 17 shows the upper 1/3 of the broken bovine incisor; (B) shows that the broken end is bonded and reset; (C) shows that the broken end is used to hang a heavy object.
  • L-DPZ hydrogel has strong viscosity.
  • Figure 19(a) shows the mixture of L-DPZ adhesive and bone powder
  • Figure 19(b) shows the implant being implanted into the mandibular defect of Bama mini pigs.
  • Photos Figure 19(c) shows a photo of the adhesive and bone powder mixture injected into the pig mandibular defect
  • Figure 19(d) shows a photo of the adhesive and bone powder mixture rinsed with water.
  • bone grafting treatment is required.
  • Conventional application of bone powder is not easy to retain. After applying L-DPZ hydrogel mixed with bone powder, it can still remain in place despite continuous erosion by water flow. .
  • L-DPZ hydrogel has good biocompatibility.
  • FIG. 24 shows the osteogenic ability of L-DPZ hydrogel (product in Example 1 and Example 2) staining of rBMSC after adding different components and culturing for 14 days, and after culturing for 21 days.
  • ARS alizarin red
  • Figure 25 shows the expression levels of osteogenic indicators BMP2 (bone morphogenetic protein 2), RUNX2 (Runt-related transcription factor 2), and ALP (alkaline phosphatase) in rBMSCs after 4/7/14 days of culture under different components. , it can be seen that L-DPZ hydrogel further enhanced the osteogenic performance.
  • BMP2 bone morphogenetic protein 2
  • RUNX2 Raster-related transcription factor 2
  • ALP alkaline phosphatase
  • L-DPZ hydrogel product in Example 2
  • a New Zealand rabbit cranial bone defect model was established, and L-DPZ hydrogel was mixed with bone powder to fill the defect site.
  • L-DPZ hydrogel was mixed with bone powder to fill the defect site.
  • simple bone powder implantation group Bone meal pellets, Geistlich AG, Switzerland
  • L-DPZ hydrogel with bone meal Bone meal particles, Geistlich, Switzerland
  • the osteogenic effect was observed after 4/8/12 weeks respectively.
  • the addition of the hydrogel not only increases the operability of the surgery, but also allows the bone powder filled in the bone defect to remain in place and remain in place without being washed away by blood or water flow.
  • the bone defect site is conducive to later osteogenesis.
  • a in Figure 27 shows the New Zealand rabbit cranial bone defect model
  • B in Figure 27 shows the state diagram after filling the L-DPZ2 hydrogel and bone powder mixed material.
  • Figure 28 is a microCT comparison chart of the blank group, bone powder group, L-DP hydrogel and bone powder group, and L-DPZ2 hydrogel and bone powder mixed group 4 weeks and 8 weeks after implantation into the parietal bone defect of rabbits. From the CT three-dimensional reconstruction results, it can be seen that the osteogenesis effect of the L-DPZ2 hydrogel and bone powder mixed group is significantly better than that of the control group, and the average bone thickness, bone density (BMD), bone volume fraction (BV/TV) and bone size The quantitative analysis results of beam number (Tb.N) showed that the osteogenic effect of the L-DPZ2 hydrogel and bone powder mixed group was significantly better than that of the control group.
  • BMD bone density
  • BV/TV bone volume fraction
  • Tb.N quantitative analysis results of beam number
  • Figure 29 shows the hard tissue section VG of the blank group, bone powder group, L-DP hydrogel and bone powder group, and L-DPZ2 hydrogel and bone powder mixed group 4 and 8 weeks after implantation into the rabbit cranial defect (Van Gieson' Spicrofuchsin) staining comparison chart.
  • the dotted line depicts the representative bone powder particles, and the dark red represents new bone formation. It can be seen that the L-DPZ2 hydrogel and bone powder mixed group promotes the formation of new bone.
  • the adhesive of commercially available medical adhesive ⁇ -cyanoacrylate (Guangzhou Baiyun Medical Adhesive Co., Ltd., medical anastomotic type) was mixed with the L-DPZ1 and L-DPZ2 hydrogels of Example 1 and Example 2.
  • a universal testing machine was used to compare the bonding shear performance between two cortical bone pieces and the tensile property test between two cortical bone pieces using different components.
  • Figure 26 is a comparison chart of osteogenic index results after 4 days of co-culture of L-DPZ hydrogel and cells at different proportions. It can be seen that the three items of L-DPZ1 and L-DPZ2 hydrogels in Example 1 and Example 2 The expression levels of osteogenic indicators, namely ALP, BMP2 and RUNX2, were significantly better than those of L-DPZ3 hydrogel in Comparative Example 2, proving that the osteogenic effects of L-DPZ1 and L-DPZ2 hydrogels were significantly better than those of L- DPZ3 hydrogel.
  • the present disclosure provides a bone adhesive, its preparation method and application, by grafting levodopa on polyvinyl alcohol, and using levodopa combined with ZIF-8 to form a multifunctional bionic ZIF-8 bone glue, Has the following advantages:
  • the present disclosure provides a bone adhesive, which uses polyvinyl alcohol (PVA), a polymer with good biocompatibility, as a carrier to construct a bone adhesive with good bone adhesion, biocompatibility, osteogenic properties, injectability, etc.
  • PVA polyvinyl alcohol
  • the new functional bone glue L-DPZ bone glue
  • L-DPZ bone glue can promote the bone bonding and bone healing process of severe bone trauma such as complex fractures and bone defects, and its preparation method is simple and widely used. Therefore, the invention provides Bone adhesives and their preparation methods have excellent practical properties and broad clinical application prospects.

Abstract

本公开提供了一种骨粘接剂、其制备方法及应用，涉及医用材料技术领域。该骨粘接剂通过在聚乙烯醇上接枝左旋多巴，并利用左旋多巴上的酚羟基结合ZIF-8形成多功能仿生ZIF-8骨胶水，利用以金属有机骨架ZIF-8为核心，整合含有大量邻苯二酚结构的左旋多巴(L-DOPA)分子的湿粘接特性，以生物相容性良好的聚合物聚乙烯醇(PVA)为载体，构建具备良好骨粘接性、生物相容性、成骨特性、可注射性等功能于一体的新型骨胶水(L-DPZ骨胶水)，能够促进复杂骨折、骨缺损等严重骨创伤的骨粘接和骨愈合过程，具备广泛的临床应用前景。

Description

一种骨粘接剂、其制备方法及应用
相关申请的交叉引用
本公开要求于2022年03月11日提交中国专利局的申请号为CN202210237275.1、名称为“一种骨粘接剂、其制备方法及应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及医用材料技术领域，具体而言，涉及一种骨粘接剂、其制备方法及应用。
背景技术
骨折是最常见的创伤之一，每年危害全球数千万人的健康。其中，严重高位骨折或粉碎性骨折会显著增加手术难度，也会增加延迟愈合和骨愈合不良的发生率。此外，由于创伤或肿瘤等导致的大块骨缺损也是一项长期的临床挑战。
传统技术如使用金属板和螺丝钉，是安全和可用的，但容易引起异物反应或松动，还需要手术更换或移除，延长了手术时间，增加了手术成本和病人的痛苦。因此，作为粘连和拼接复杂骨折的辅助工具的骨粘接剂/骨胶水可能是一种极具吸引力的方法，有可能改变治疗高度粉碎性骨折的手术策略。
近年来，聚甲基丙烯酸甲酯(PMMA)、磷酸钙(CPC)、氰基丙烯酸酯(CA)等骨粘接剂被广泛使用。但是，目前的产品仍然存在严重的缺陷，主要表现在以下方面：(1)PMMA与骨的固有粘附性很小，会导致严重的热和化学坏死，以及有毒单体和不可生物吸收；(2)CPC的机械性能较差，暴露在体液中容易塌陷，这会加剧骨骼的破坏；(3)以CA为代表的胶粘剂在体内降解缓慢，不能及时为新生组织生长提供空间。
因此，有必要开发一种具备优异湿粘接性能、生物相容性、可操作性、降解性能的新型骨粘接剂，以便在潮湿的生物环境中实现对骨折和缺损的充分和稳定的固定。此外，临床上理想的骨粘接剂还应该表现出生物可吸收性和骨整合能力，以促进新骨再生，及时恢复结构和机械完整性。
发明内容
本公开提供一种骨粘接剂的制备方法，包括：利用聚乙烯醇和左旋多巴反应得到L-DP聚合物，将L-DP聚合物与ZIF-8反应，以使L-DP聚合物上的酚羟基与锌离子螯合。
可选地，所述L-DP聚合物与ZIF-8的质量比为100:1-2。
可选地，控制聚乙烯醇和左旋多巴用量的摩尔比为4-7:1。
可选地，所述L-DP聚合物是利用分子量为85-124kDa的聚乙烯醇与左旋多巴反应而得。
可选地，所述L-DP聚合物的制备方法包括：将聚乙烯醇溶解之后，在催化剂存在的条件下与左旋多巴反应，将反应后的溶液制备形成凝胶。
可选地，所述凝胶的制备过程包括：将反应后的溶液透析2-3天，再进行旋蒸、冻干。
可选地，所述聚乙烯醇和左旋多巴的反应温度为75-85℃，反应时间为15-20h。
可选地，所述聚乙烯醇和左旋多巴的反应温度为78-82℃，反应时间为12-18h。
可选地，将所述聚乙烯醇溶解是在加热的条件下溶解于有机溶剂，有机溶剂为二甲基亚砜。
可选地，反应过程中通入保护气体。
可选地，所采用的催化剂为硫酸氢钠水合物。
可选地，硫酸氢钠水合物与聚乙烯醇的质量比为3-4:1。
可选地，将所述凝胶复溶形成的水溶液与ZIF-8形成的水溶液反应，反应时间为0.5-1h。
可选地，将所述凝胶与ZIF-8反应后的溶液制备形成水凝胶。
可选地，将所述凝胶与ZIF-8反应后，利用伽马射线照射消毒并使原料进一步交联成胶。
可选地，所述凝胶形成的水溶液的浓度为150-250mg/mL，ZIF-8形成的水溶液的浓度为25-100mg/mL。
本公开还提供一种骨粘接剂，通过上述制备方法制备而得。
可选地，骨粘接剂为水凝胶的形态。
本公开还提供上述骨粘接剂在制备骨组织修复材料中的应用。
本公开还提供一种生物医用材料，通过上述制备方法制备而得。
本公开还提供所述骨粘接剂或者所述生物医用材料，用于骨组织粘接、骨组织修复和/或骨组织愈合的用途。
本公开还提供一种治疗与骨创伤相关疾病的方法，包括：
向有此需要的受试者施用所述骨粘接剂或者所述生物医用材料。
可选地，所述与骨创伤相关疾病包括骨折或骨缺损中的至少一种。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为ZIF-8纳米颗粒的扫描电镜图；
图2为L-DP凝胶电镜下形成多孔的表面形貌；
图3为L-DPZ水凝胶电镜下形成多孔的表面形貌；
图4为L-DPZ水凝胶的元素分析图；
图5为L-DP和L-DPZ水凝胶的傅立叶红外光谱测试(FT-IR)图；
图6为L-DP和L-DPZ水凝胶的X线光电子能谱(XPS)图；
图7为两块不同颜色L-DPZ水凝胶切断后结合并发生自愈合行为图；
图8为L-DPZ水凝胶在固定角频率(10rad/s)、37℃下进行应变振幅扫描试验(1-1000％)结果；
图9为在恒角频率(10rad/s)下进行L-DPZ水凝胶连续循环应变试验(5％应变→600％应变→5％应变)结果；
图10为L-DPZ水凝胶剪切变稀测试结果；
图11为利用哑铃测试L-DPZ水凝胶对牛骨片的粘接强度；
图12为利用人测试L-DPZ水凝胶对牛骨片的粘接强度；
图13为L-DPZ水凝胶对截断的牛胫骨的粘接作用图；
图14为骨片剪切力测试图，其中，a为骨片剪切力测试方法示意图，b为骨片剪切力测试数据表征)；
图15为骨块拉伸力测试图，其中a为骨块拉伸力测试方法示意图，b为骨块拉伸力测试数据表征)；
图16为PVA、L-DP及L-DPZ2水凝胶对聚四氟乙烯和玻璃的粘接能力测试图，其中a和b分别为PVA、L-DP及L-DPZ2水凝胶对聚四氟乙烯粘接能力测试方法示意图和粘接能力数据表征，c和d则为PVA、L-DP及L-DPZ2水凝胶对玻璃的粘接能力测试方法示意图和粘接能力数据表征；
图17为L-DPZ水凝胶对牛牙碎片的粘接能力测试图；
图18为L-DPZ水凝胶对脱位猪牙粘接复位能力；
图19为L-DPZ水凝胶与骨粉混合后的粘接性能测试图；
图20为L-DP及L-DPZ水凝胶与骨粉混和物的压缩强度及三点弯曲强度测试图；
图21为L-DP和L-DPZ水凝胶的体外降解速率检测结果；
图22为L-DP和L-DPZ水凝胶的体内降解速率检测结果；
图23为rBMSC的CCK8活性测试结果图；
图24为L-DPZ水凝胶对rBMSC的成骨性能ALP染色及ARS染色图；
图25为L-DPZ水凝胶对rBMSC的成骨性能qPCR测试结果图；
图26为不同比例L-DPZ水凝胶成骨性能测试结果对比图；
图27为L-DPZ水凝胶在动物体内的成骨能力测试手术图；
图28为空白组、单纯骨粉组、L-DP水凝胶与骨粉混和组以及L-DPZ水凝胶与骨粉混合组4及8周后的新西兰兔体内成骨效果micro-CT对比图；
图29为为空白组、单纯骨粉组、L-DP水凝胶与骨粉混合组以及L-DPZ水凝胶与骨粉混合组4及8周后的新西兰兔体内成骨效果VG染色对比图。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本公开一实施方式提供一种骨粘接剂的制备方法，包括以下步骤：
S1、L-DP聚合物的合成
利用聚乙烯醇和左旋多巴反应得到L-DP聚合物，利用聚乙烯醇上的氢键将左旋多巴引入聚乙烯醇的长链上，使左旋多巴接枝在聚乙烯醇上得到L-DP聚合物。
可选地，左旋多巴即左旋3，4-二羟基-苯丙氨酸(L-DOPA)具有良好的湿粘接性潜能。
在实际操作过程中，L-DP聚合物的制备方法包括：将聚乙烯醇溶解之后，在催化剂存在的条件下与左旋多巴反应，将反应后的溶液制备形成凝胶，以便后续引入ZIF-8。
L-DP聚合物是利用分子量为85-124kDa(例如90-120kDa、95-115kDa或100-110kDa，诸如85kDa、90kDa、95kDa、100kDa、105kDa、110kDa、115kDa、120kDa，或者任意两个端点值之间的数值区间)的聚乙烯醇与左旋多巴反应而得，且控制聚乙烯醇和左旋多巴的摩尔比为4-7:1，例如4:1、5:1、6:1、7:1等，或者任意两个端点值之间的数值区间。
可选地，将聚乙烯醇溶解是在加热的条件下溶解于有机溶剂。在可选的实施方式中，有机溶剂选择二甲基亚砜(DMSO)为宜。
可选地，所采用的催化剂为硫酸氢钠水合物为宜。催化剂的用量不限，在一些实施方式中可以控制硫酸氢钠水合物与聚乙烯醇的质量比为3-4：1，诸如3.1：1、3.2：1、3.3：1、3.4：1、3.5：1、3.6：1、3.7：1、3.8：1、3.9：1或4.0：1，或者任意两个端点值之间的数值区间。
可选地，聚乙烯醇和左旋多巴的反应温度为75-85℃，反应时间为15-20h。可选地，聚乙烯醇和左旋多巴反应温度为78-82℃，反应时间为12-18h。通过进一步控制聚乙烯醇和左旋多巴的反应温度和时间以使二者反应完全，提高左旋多巴的接枝率。
可选地，聚乙烯醇和左旋多巴的反应温度可以为77-83℃、79-82℃或80-81℃，诸如76℃、77℃、78℃、79℃、80℃、81℃、82℃、83℃、84℃或85℃等，或者任意两个端点值之间的数值区间，也可以为以上相邻温度值之间的任意值；反应时间可以为例如14-19h、15-18h或16-17h，诸如12h、13h、14h、15h、16h、17h、18h等，或者任意两个端点值之间的数值区间，也可以为以上相邻时间值之间的任意值。
在可选的实施方式中，反应过程中通入保护气体，以防止L-DOPA在反应过程中氧化。可选地，保护气体的种类不限，可以为氮气或其他惰性气体。
在一些实施方式中，凝胶的制备过程包括：将反应后的溶液透析2-3天，再进行旋蒸、冻干。在其他实施方式中，旋蒸可以替换为其他蒸发形式，能够去除大部分水分即可；冻干也可以替换为其他形式的干燥形式。
S2、L-DPZ水凝胶的制备
将L-DP聚合物与ZIF-8反应，利用ZIF-8中的锌离子与左旋多巴中形成金属酚类配位键，引入锌离子。可选地，ZIF-8是以锌离子为连接体，可持续释放Zn 2+，可以在成骨、血管生成和抗菌过程中发挥积极作用。
在实际操作过程中，将凝胶复溶形成的水溶液与ZIF-8形成的水溶液反应，反应时间为0.5-1h；将凝胶与ZIF-8反应后的溶液制备形成水凝胶。可选地，将凝胶与ZIF-8反应后，利用伽马射线照射(15Gmy)照射8h消毒并使二者进一步交联成胶。
在其他实施方式中，最终的产品L-DPZ也可以不以水凝胶的形式存在。
在可选的实施方式中，控制凝胶和ZIF-8的质量比为100:1-2，例如100:1.1、100:1.3、100:1.4、100:1.5、100:1.6、100:1.7、100:1.8、100:1.9或100:2.0，或上述任意两个端点的区间值。凝胶形成的水溶液的浓度为150-250mg/mL，例如160-240mg/mL、170-230mg/mL或180-220mg/mL，诸如150mg/mL、160mg/mL、170mg/mL、180mg/mL、190mg/mL、200mg/mL、210mg/mL、220mg/mL、230mg/mL、240mg/mL或250mg/mL，，或上述任意两个端点的区间值。ZIF-8形成的水溶液的浓度为25-100mg/mL，例如30-90mg/mL、40-80mg/mL或50-70mg/mL，诸如26mg/mL、30mg/mL、40mg/mL、50mg/mL、60mg/mL、70mg/mL、80mg/mL、90mg/mL或100mg/mL，或上述任意两个端点的区间值。通过进一步控制ZIF-8的引入量，可以使产品具备优异的骨粘接性能的前提下，保证产品的毒性满足要求，若ZIF-8的 引入量过大则会导致毒性过强；若ZIF-8的引入量过小则会显著降低产品的骨粘接性能。
本公开一实施方式还提供一种骨粘接剂，可以通过以上制备方法制备而得。由于该骨粘接剂具备非常优异的骨粘接性能且生物相容性较好，可以在制备骨组织修复材料中得到应用，以促进复杂骨折、骨缺损等严重骨创伤的骨粘接和骨愈合过程，具备广泛的临床应用前景。
在一些实施方式中，在聚乙烯醇和左旋多巴形成的L-DP聚合物与ZIF-8的质量比为100:1-2。
本公开一实施方式提供一种生物医用材料，通过上述制备方法制备而得。
本公开一实施方式还提供骨粘接剂或者生物医用材料，用于骨组织粘接、骨组织修复和/或骨组织愈合的用途。
本公开一实施方式还提供一种治疗与骨创伤相关疾病的方法，包括：
向有此需要的受试者施用骨粘接剂或者生物医用材料。
在一些实施方式中，与骨创伤相关疾病包括骨折或骨缺损中的至少一种。
本公开提供了一种骨粘接剂及其制备方法，提升了粘接剂的骨粘接性、生物相容性和成骨特性。
本公开通过在聚乙烯醇上接枝左旋多巴，并利用左旋多巴上的酚羟基结合ZIF-8形成多功能仿生ZIF-8骨胶水，利用以金属有机骨架ZIF-8为核心，整合含有大量邻苯二酚结构的左旋多巴(L-DOPA)分子的湿粘接特性，以生物相容性良好的聚合物聚乙烯醇(PVA)为载体，构建具备良好骨粘接性、生物相容性、成骨特性、可注射性等功能于一体的新型骨胶水(L-DPZ骨胶水)，能够促进复杂骨折、骨缺损等严重骨创伤的骨粘接和骨愈合过程，具备广泛的临床应用前景。
实施例
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
本实施例提供一种骨粘接剂的制备方法，包括以下步骤：
(1)L-DP聚合物的合成
称取60mmoL的分子量为85-124kDa的PVA，在100℃条件下完全溶解于120mL的DMSO中。继而加入10g硫酸氢钠水合物，完全溶解后将温度降至80℃，然后加入10mmoL左旋多巴(L-DOPA)，并在烧瓶中充入N 2保护，防止L-DOPA在反应过程中氧化。反应12小时后，将反应所得溶液透析3天。将透析所得溶液在悬转蒸发仪蒸除大部分水分，得到40mL左右液体，最终在冻干机中冻干2日，得到干燥疏松状态水凝胶。
(2)L-DPZ水凝胶的制备
将80mg ZIF-8颗粒在超声震荡下溶于2mL去离子水中。
将步骤(1)中冻干状态水凝胶称重，以200mg/mL的浓度溶于去离子水中，同时加入ZIF-8，使L-DP聚合物：ZIF8质量比达到100:1；用搅拌子完全搅拌均匀反应1h，反应后利用伽马射线照射(15Gmy)照射8h消毒并使二者进一步交联成胶，以L-DPZ1表示。
实施例2
与实施例1的区别仅在于：L-DP聚合物：ZIF8质量比达到100:2，产品以L-DPZ2表示。
实施例3
本实施例提供一种骨粘接剂的制备方法，包括以下步骤：
(1)L-DP聚合物的合成
称取60mmoL的分子量为89-98kDa的PVA，在100℃条件下完全溶解于110mL的DMSO中。继而加入8g硫酸氢钠水合物，完全溶解后将温度降至75℃，然后加入12mmoL左旋多巴(L-DOPA)，并在烧瓶中充入N 2保护，防止L-DOPA在反应过程中氧化。反应20小时后，将反应所得溶液透析3天。将透析所得溶液在悬转蒸发仪蒸除大部分水分，得到40mL左右液体，最终在冻干机中冻干2日，得到干燥疏松状态水凝胶。
(2)L-DPZ水凝胶的制备
将50mg ZIF-8颗粒在超声震荡下溶于1mL去离子水中。
将步骤(1)中冻干状态水凝胶称重，以150mg/mL的浓度溶于去离子水中，同时加入ZIF-8，使L-DP：ZIF8质量比达到100:1，用搅拌子完全搅拌均匀反应0.5h，反应后利用伽马射线照射(15Gmy)照射8h消毒并使二者进一 步交联成胶。
实施例4
本实施例提供一种骨粘接剂的制备方法，包括以下步骤：
(1)L-DP聚合物的合成
称取75mmoL的分子量为13-23kDa的PVA，在100℃条件下完全溶解于140mL的DMSO中。继而加入10g硫酸氢钠水合物，完全溶解后将温度降至85℃，然后加入15mmoL左旋多巴(L-DOPA)，并在烧瓶中充入N 2保护，防止L-DOPA在反应过程中氧化。反应15小时后，将反应所得溶液透析2天。将透析所得溶液在悬转蒸发仪蒸除大部分水分，得到40mL左右液体，最终在冻干机中冻干2日，得到干燥疏松状态水凝胶。
(2)L-DPZ水凝胶的制备
将50-100mg ZIF-8颗粒在超声震荡下溶于1-2mL去离子水中。
将步骤(1)中冻干状态水凝胶称重，以250mg/mL的浓度溶于去离子水中，同时加入ZIF-8，使L-DP：ZIF8质量比达到100:2，用搅拌子完全搅拌均匀反应1h，反应后利用伽马射线照射(15Gmy)照射8h消毒并使二者进一步交联成胶。
对比例1
将市售的医用粘接剂α-氰基丙烯酸酯(广州白云医用胶有限公司，医用吻合型)的粘接剂与来自实施例1的L-DPZ1水凝胶以及来自实施例2的L-DPZ2水凝胶的粘接性能进行对比。
对比例2
与实施例1的区别仅在于：L-DP：ZIF8质量比为100:4，用L-DPZ3水凝胶表示。
试验例1
材料表征：
测试实施例1中所采用的ZIF-8纳米颗粒的扫描电镜图，如图1所示；测试实施例1中L-DP及L-DPZ水凝胶在扫描电镜下形成多孔的表面形貌图，如图2和图3所示；采用德国ZEISS GeminiSEM 300对实施例1中L-DPZ水凝胶进行元素分析，结果如图4所示。
从图1可以看出，ZIF8大小均匀，基本在80-100nm之间，无过大或过小颗粒。
从图2和图3所示，L-DP及L-DPZ两种凝胶均呈现出多孔的形貌，且材料的均匀度较好。
从图4可以看出，锌离子在水凝胶中呈现出均匀分布的状态。
从图5可以看出，加入ZIF8后羟基和酚羟基发生了消耗。
从图6可以看出，加入ZIF-8后L-DPZ1和L-DPZ2较L-DP而言多了Zn-O键，表面Zn 2+的成功引入。
试验例2
测试实施例2中得到水凝胶的流变性能，具体如下：
(1)如图7所示，将两块L-DPZ水凝胶分别染为红蓝两色，并剪切为两半，将蓝的一半与红的一半相结合，半小时后，观察到二者发生自愈合。
(2)对L-DPZ水凝胶在固定角频率(10rad/s)、37℃下进行应变振幅扫描试验(1-1000％)(测试仪器为：旋转流变仪(安东帕集团，奥地利))。结果如图8所示，水凝胶在应变100％以前的G'和G”几乎保持不变，这表明L-DPZ水凝胶可以维持相对较大的弹性变形。当应变进一步增加时，G'和G”值急剧下降，且在≈600％的应变时趋于相等，表明当水凝胶网络达到临界应变值时，水凝胶网络破裂，转变为溶胶状态。
(3)在恒角频率(10rad/s)下进行连续循环应变试验(5％应变→600％应变→5％应变)(测试仪器为：旋转流变仪(安东帕集团，奥地利))。实验结果如图9所示，在超过临界应变值600％应变时，水凝胶转变为溶胶状态(G'<G”)。而一旦应变恢复到5％，凝胶再次发生变化，G'和G”值立即恢复到初始值，没有任何损失，表明L-DPZ水凝胶具备良好的自愈合特性。
(4)在37℃下测定了水凝胶的剪切变稀特性，(测试仪器为：旋转流变仪(安东帕集团，奥地利))，如图10所示，水凝胶的黏度对着剪切速率的增加而降低，即水凝胶具备良好的剪切变稀特性，证明L-DPZ水凝胶良好的可注射性。
试验例3
粘接性能测试：
(1)使用L-DPZ2水凝胶(实施例2中产品)粘接两块牛骨片后，如图11和图12所示，不仅能够承重60kg的哑铃，还可以承受两个体重约60kg(合计约120kg)的成年人的重量，展现了该骨粘接剂超强的粘接性能。
(2)取一段牛胫骨，从中截断，再用L-DPZ2水凝胶(实施例2中产品)将其粘接复位，在两端悬挂9.07kg重物仍不会断裂，测试过程如图13所示。
(3)通过万能力学测试仪定量测试了PVA、L-DP、L-DPZ1(三者均来自实施例1中产品)和L-DPZ2(实施例2中产品)水凝胶的粘接能力。图14、图15分别为不同组分对两块皮质骨片之间粘接剪切性能以及两块皮质骨块之间拉伸性能测试，可以看出，不同配比的L-DPZ水凝胶可以达到较好的机械性能。
(4)测试PVA、L-DP、L-DPZ1(三者均来自实施例1中产品)和L-DPZ2(实施例2中产品)水凝胶对于其他不同粘接界面的粘接能力，如图16所示，分别测试了水凝胶对聚四氟乙烯和玻璃的粘接强度，结果均表明L-DPZ水凝胶具备优异的粘接性能。
(5)将一颗牛切牙上1/3水平磨断后再用L-DPZ2水凝胶(实施例2中产品)将其粘接复位，并在断端打孔悬吊5kg重物，如图15所示，可以看出本公开实施例所制备的L-DPZ水凝胶展现相当好的粘接性能。
图17中(A)表示上1/3磨断的牛切牙；(B)表示将断端粘接复位；(C)表示利用断端悬挂重物。
(6)将猪下颌右侧中切牙拔出后，在拔牙窝内注射水凝胶，再将牙齿粘接复位，并在牙冠处打孔悬吊1000g重物，如图18所示，可以看出本公开实施例所制备的L-DPZ水凝胶展现相当好的粘接性能。
图18中①表示在拔出牙的拔牙窝内注射水凝胶；②将拔除的牙齿放入拔牙窝内复位；③表示利用牙冠部悬挂重物。
(7)L-DPZ水凝胶较强粘性，使L-DPZ2水凝胶(实施例2中产品)与骨粉混合，如图19所示，水凝胶混合骨粉后，赋予骨粉之间粘附性能，增强了使用骨粉时的可操作性能，其中图19(a)示出为L-DPZ粘接剂和骨粉混合物，图19(b)为将种植体植入巴马小型猪下颌骨缺损处照片，图19(c)示出为将粘接剂和骨粉混合物注射在猪下颌骨缺损处照片，图19(d)示出为用水冲洗粘接剂和骨粉混合物照片。此外，建立猪下颌骨植入种植体后暴露模型，需行植骨治疗，常规应用骨粉不易固位，应用L-DPZ水凝胶混合骨粉后，尽管在水流持续冲刷下，仍能保持原位。
(8)将L-DP、L-DPZ、L-DPZ2(实施例1、2中产品)与骨粉混合，如图20所示，水凝胶与骨粉混合后，获得了较强的压缩强度及三点弯曲强度，并随着ZIF8含量的提高，机械性能也相应提高。
试验例4
(1)测试L-DPZ水凝胶(实施例1和实施例2中产品)的体外降解速率，将水凝胶冻干称重，用m 1表示，然后将不同配比的水凝胶置于PBS溶液中，在不同的时间点(2h、4h、6h、8h、24h和3d)取出水凝胶，冻干后再次称重，用m 2表示，计算水凝胶不同时间的降解率Q＝(m 1-m 2)/m1×100％，检测结果如图21所示，不同配比的L-DPZ水凝胶降解速率均较稳定，随着时间的增加逐步缓慢降解，从而为新生骨组织提供引导和生长空间。
(2)测试L-DPZ水凝胶(实施例1和实施例2中产品)的体内降解速率，将水凝胶冻干称重，用m 1表示，然后将不同配比的水凝胶置于SD大鼠(雄性，5周龄)皮下，在不同的时间点(1周、2周、4周)取出水凝胶，冻干后再次称重，用m 2表示，计算水凝胶不同时间的降解率Q＝(m 1-m 2)/m1×100％，检测结果如图22所示，不同配比的L-DPZ水凝胶降解速率均较稳定，随着时间的增加逐步缓慢降解，从而为新生骨组织提供引导和生长空间。
试验例5
测试L-DPZ水凝胶(实施例1和实施例2中产品)的体外生物安全性，通过CCK8测试不同组分对rBMSC(大鼠骨髓间充质干细胞)活性的影响，如图23所示，可以看出L-DPZ水凝胶具有良好的生物相容性。
试验例6
测试L-DPZ水凝胶(实施例1和实施例2中产品)的成骨能力，图24为加入不同组分培养14天后的rBMSC的ALP(碱性磷酸酶)染色，以及培养21天后的ARS(茜素红)染色，染色越深表示成骨能力越强。
图25中分别为不同组分下培养4/7/14天后，rBMSC中成骨指标BMP2(骨形态发生蛋白2)、RUNX2(Runt相关转录因子2)、ALP(碱性磷酸酶)的表达水平，可以看到，L-DPZ水凝胶进一步增强了成骨性能。
试验例7
为了评估L-DPZ水凝胶(实施例2中产品)在动物体内的成骨能力，建立了新西兰兔颅顶骨缺损模型，采用将L-DPZ水凝胶与骨粉混合的形式填入缺损部位，并与空白组、单纯植入骨粉组(
Figure PCTCN2023070599-appb-000001
骨粉颗粒，盖氏公司，瑞士) 以及L-DPZ水凝胶与骨粉(
Figure PCTCN2023070599-appb-000002
骨粉颗粒，盖氏公司，瑞士)混合组做对比，分别在4/8/12周后，观察成骨效果。与单纯骨粉相比，加入该水凝胶不仅增加了手术的可操作性，还可使填入骨缺损部位的骨粉保持在原位不移动，不被血液、水流的冲洗下仍能够持续存留于骨缺损部位，利于后期成骨作用。
图27中A表示新西兰兔颅顶骨缺损模型；图27中B表示填入L-DPZ2水凝胶与骨粉混合材料后的状态图。
图28为空白组、骨粉组、L-DP水凝胶与骨粉组和L-DPZ2水凝胶与骨粉混合组植入兔颅顶骨缺损后4周及8周microCT对比图。CT三维重建结果可以看出，L-DPZ2水凝胶与骨粉混合组的成骨效果明显优于对照组，且平均骨厚度、骨密度(BMD)、骨体积分数(BV/TV)及骨小梁数(Tb.N)定量分析结果均表明L-DPZ2水凝胶与骨粉混合组的成骨效果显著优于对照组。
图29为空白组、骨粉组、L-DP水凝胶与骨粉组和L-DPZ2水凝胶与骨粉混合组植入兔颅顶缺损后4周及8周的硬组织切片VG(Van Gieson'spicrofuchsin)染色对比图。虚线描出了代表性骨粉颗粒，深红代表新骨形成，可以看出L-DPZ2水凝胶与骨粉混合组，促进了新骨的形成。
试验例8
将市售的医用粘接剂α-氰基丙烯酸酯(广州白云医用胶有限公司，医用吻合型)的粘接剂与实施例1和实施例2的L-DPZ1和L-DPZ2水凝胶的粘接性能进行对比，利用万能测试机分别比较了不同组分对两块皮质骨片之间粘接剪切性能以及两块皮质骨块之间拉伸性能测试。
可以看出，实施例1和实施例2的L-DPZ1和L-DPZ2水凝胶的粘接强度明显优于市售的医用粘接剂α-氰基丙烯酸酯(如图14、图15所示)。
试验例9
将实施例1和实施例2的L-DPZ1和L-DPZ2水凝胶的成骨性能与对比例2(即L-DP：ZIF8质量比为100:4)进行对比，比较不同比例的成骨性能。
图26为不同比例L-DPZ水凝胶与细胞共培养4天后的成骨指标结果对比图，可以看出，实施例1和实施例2的L-DPZ1和L-DPZ2水凝胶的三项成骨指标，即ALP、BMP2和RUNX2的表达水平显著优于对比例2中L-DPZ3水凝胶的表达水平，证明L-DPZ1和L-DPZ2水凝胶的成骨效果明显优于L-DPZ3水凝胶。
综上，本公开提供一种骨粘接剂、其制备方法及应用，通过在聚乙烯醇上接枝左旋多巴，并利用左旋多巴结合ZIF-8形成多功能仿生ZIF-8骨胶水，具备以下优点：
(1)具有可靠的力学性能和骨粘接性，能够牢固粘接骨碎片，复位骨折部位；
(2)具备良好的成骨能力，有效促进骨重建；
(3)良好的生物相容性，具备进一步应用于医学领域的前提；
(4)良好的自愈合性能和可注射性，具备良好的形状适配能力，能够适应不同形状的缺损和骨碎片；
(5)适当的降解速率，为新生骨组织提供引导和生长空间。
以上仅为本公开的可选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
本公开提供了一种骨粘接剂，以生物相容性良好的聚合物聚乙烯醇(PVA)为载体，构建具备良好骨粘接性、生物相容性、成骨特性、可注射性等功能于一体的新型骨胶水(L-DPZ骨胶水)，能够促进复杂骨折、骨缺损等严重骨创伤的骨粘接和骨愈合过程，且其制备方法及简便，应用广泛，因此本公开提供的骨粘接剂及其制备方法均具备优异的实用性能和广阔的临床应用前景。

Claims (14)

  1. 一种骨粘接剂的制备方法，其特征在于，包括：利用聚乙烯醇和左旋多巴反应得到L-DP聚合物，将所述L-DP聚合物与ZIF-8反应，以使所述L-DP聚合物上的酚羟基与锌离子螯合。
  2. 根据权利要求1所述的制备方法，其特征在于，所述L-DP聚合物与所述ZIF-8的质量比为100:1-2。
  3. 根据权利要求2所述的制备方法，其特征在于，控制聚乙烯醇和左旋多巴用量的摩尔比为4-7:1。
  4. 根据权利要求3所述的制备方法，其特征在于，所述L-DP聚合物是利用分子量为85-124kDa的聚乙烯醇与左旋多巴反应而得；
    优选地，所述L-DP聚合物的制备方法包括：将聚乙烯醇溶解之后，在催化剂存在的条件下与左旋多巴反应，将反应后的溶液制备形成凝胶；
    优选地，所述凝胶的制备过程包括：将反应后的溶液透析2-3天，再进行旋蒸、冻干。
  5. 根据权利要求4所述的制备方法，其特征在于，所述聚乙烯醇和所述左旋多巴的反应温度为78-82℃，反应时间为12-18h；
    优选地，所述聚乙烯醇和所述左旋多巴的反应温度为78-82℃，反应时间为16-18h；
    优选地，将所述聚乙烯醇溶解是在加热的条件下溶解于有机溶剂，所述有机溶剂为二甲基亚砜；
    优选地，反应过程中通入保护气体。
  6. 根据权利要求4或5所述的制备方法，其特征在于，所采用的催化剂为硫酸氢钠水合物；
    优选地，硫酸氢钠水合物与聚乙烯醇的质量比为3-4:1。
  7. 根据权利要求4-6中任一项所述的制备方法，其特征在于，将所述凝胶复溶形成的水溶液与ZIF-8形成的水溶液反应，反应时间为0.5-1h；
    优选地，将所述凝胶与所述ZIF-8反应后的溶液制备形成水凝胶；
    更优选地，将所述凝胶与所述ZIF-8反应后，利用伽马射线照射消毒并使原料进一步交联成胶。
  8. 根据权利要求7所述的制备方法，其特征在于，所述凝胶形成的水溶液的浓度为150-250mg/mL，所述ZIF-8形成的水溶液的浓度为25-100mg/mL。
  9. 一种骨粘接剂，其特征在于，通过权利要求1-8中任一项所述的制备方法制备而得；
    优选地，所述骨粘接剂为水凝胶的形态。
  10. 权利要求9中所述的骨粘接剂在制备骨组织修复材料中的应用。
  11. 一种生物医用材料，其特征在于，通过权利要求1-8中任一项所述的制备方法制备而得。
  12. 根据权利要求9所述的骨粘接剂或者根据权利要求11所述的生物医用材料，用于骨组织粘接、骨组织修复和/或骨组织愈合的用途。
  13. 一种治疗与骨创伤相关疾病的方法，包括：
    向有此需要的受试者施用根据权利要求9所述的骨粘接剂或者根据权利要求11所述的生物医用材料。
  14. 根据权利要求13所述的方法，其特征在于，所述与骨创伤相关疾病包括骨折或骨缺损中的至少一种。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102596275A (zh) * 2009-09-04 2012-07-18 亚洲大学校产学协力团 用于组织粘合剂的原位成型水凝胶及其生物医学用途
CN109705369A (zh) * 2018-12-27 2019-05-03 暨南大学 一种海藻酸钠-多巴胺/聚乙烯醇水凝胶及其制备方法与应用
US20210106718A1 (en) * 2019-10-14 2021-04-15 Board Of Regents, The University Of Texas System Mussel inspired nancomposite adhesives for biomedical applications
US20210163797A1 (en) * 2019-12-03 2021-06-03 Massachusetts Institute Of Technology Body fluid resistant tissue adhesives
CN114470320A (zh) * 2022-03-11 2022-05-13 重庆医科大学 一种骨粘接剂、其制备方法及应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110368527A (zh) * 2018-07-18 2019-10-25 尹静波 一种温敏粘附性脱钙骨复合组织工程支架及制备方法
EP4181962A1 (en) * 2020-07-14 2023-05-24 The Regents of University of California Dopa modified gelatin for wound healing and methods of making the same
CN113679891B (zh) * 2021-05-21 2022-08-30 四川大学 一种zif-8载药水凝胶促成骨支架、制备方法及应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102596275A (zh) * 2009-09-04 2012-07-18 亚洲大学校产学协力团 用于组织粘合剂的原位成型水凝胶及其生物医学用途
CN109705369A (zh) * 2018-12-27 2019-05-03 暨南大学 一种海藻酸钠-多巴胺/聚乙烯醇水凝胶及其制备方法与应用
US20210106718A1 (en) * 2019-10-14 2021-04-15 Board Of Regents, The University Of Texas System Mussel inspired nancomposite adhesives for biomedical applications
US20210163797A1 (en) * 2019-12-03 2021-06-03 Massachusetts Institute Of Technology Body fluid resistant tissue adhesives
CN114470320A (zh) * 2022-03-11 2022-05-13 重庆医科大学 一种骨粘接剂、其制备方法及应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIU YANHUA, ZHU ZHOU, PEI XIBO, ZHANG XIN, CHENG XINTING, HU SHANSHAN, GAO XIAOMENG, WANG JIAN, CHEN JUNYU, WAN QIANBING: "ZIF-8-Modified Multifunctional Bone-Adhesive Hydrogels Promoting Angiogenesis and Osteogenesis for Bone Regeneration", APPLIED MATERIALS & INTERFACES, AMERICAN CHEMICAL SOCIETY, US, vol. 12, no. 33, 19 August 2020 (2020-08-19), US , pages 36978 - 36995, XP093090459, ISSN: 1944-8244, DOI: 10.1021/acsami.0c12090 *
刘蓉瑾 (LIU, RONGJIN): "含儿茶酚基聚合物的制备及其性能研究 (Study on Preparation and Properties of Catechol-Based Polymers)", 中国优秀硕士学位论文全文数据库 工程技术I辑 (ENGINEERING SCIENCE AND TECHNOLOGY I, CHINESE MASTER’S THESES FULL-TEXT DATABASE), no. 12, 15 December 2015 (2015-12-15) *

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