WO2018019208A1 - 新的骨材料替代合成配方、制备方法和使用方法 - Google Patents

新的骨材料替代合成配方、制备方法和使用方法 Download PDF

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WO2018019208A1
WO2018019208A1 PCT/CN2017/094133 CN2017094133W WO2018019208A1 WO 2018019208 A1 WO2018019208 A1 WO 2018019208A1 CN 2017094133 W CN2017094133 W CN 2017094133W WO 2018019208 A1 WO2018019208 A1 WO 2018019208A1
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bone
bone cement
material composite
composite particles
copolymer
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PCT/CN2017/094133
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English (en)
French (fr)
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黄棨麟
黄仲廉
赖景然
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薪创生命科技有限公司
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Priority to US16/319,872 priority Critical patent/US11110205B2/en
Priority to EP17833511.3A priority patent/EP3488876B8/en
Publication of WO2018019208A1 publication Critical patent/WO2018019208A1/zh

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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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
    • 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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • 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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/0047—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L24/0073—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material with a macromolecular matrix
    • A61L24/0084—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material with a macromolecular matrix containing fillers of phosphorus-containing inorganic compounds, e.g. apatite
    • 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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/02—Surgical adhesives or cements; Adhesives for colostomy devices containing inorganic 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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/06—Surgical adhesives or cements; Adhesives for colostomy devices containing 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
    • A61L27/02—Inorganic materials
    • A61L27/12—Phosphorus-containing materials, e.g. apatite
    • 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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/12—Esters of monohydric alcohols or phenols
    • C08F220/14—Methyl esters, e.g. methyl (meth)acrylate
    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • 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
    • 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
    • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus

Definitions

  • the invention belongs to the field of bone material application, in particular, the invention relates to a new bone material replacement synthetic formula, a preparation method and a using method.
  • PMMA Polymethyl methacrylate
  • bone cement has been widely used as a clinical bone tissue repair material for many years. Although it has good physical and chemical properties and mechanical properties, its hydrophilic properties are poor and the curing temperature is high, resulting in bone. The compatibility between cement material and bone tissue is poor, it is difficult to form a good combination with bone tissue, and the viscosity of bone cement is high, which is not conducive to syringe injection.
  • PMMA has a high curing temperature (up to 120 ° C), insufficient bone binding, and looseness, dislocation, and even shedding between bone tissue (especially osteoporosis). These defects can easily lead to various postoperative complications. disease. After the implantation of the current PMMA bone cement, a thin connective tissue is formed on the surface of the bone cement, which will impair the direct force transmission between the implant and the bone, which will eventually lead to premature loosening of the prosthesis.
  • U.S. Patent No. 5,642,215 B1 discloses the addition of 4-MET monomer (4-methacryloyloxy trimellitic anhydride) or 4-META (4-methacryloyloxyphthalic anhydride) and calcium phosphate to bone cement, in particular Hydroxyapatite. These bone cement formulations have unreacted monomeric additives that may adversely affect the body.
  • U.S. Patent No. 8,834,845 B2 discloses a bioactive bone cement which, upon implantation, provides a bioactive surface and promotes the formation of calcium phosphate on the surface of the bone cement.
  • CPCs Calcium phosphate cements
  • CPCs have excellent biocompatibility, bioactivity and osteoconductivity.
  • CPCs without any additives usually have poor injection properties due to liquid-solid phase separation; in addition, they are weakly cohesive and tend to collapse at the beginning of contact with blood or biological fluids; and poor mechanical properties, including toughness, brittleness, and Reliability further limits its further application.
  • a bone material composite particle comprising a copolymer of methyl hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA), and coated a calcium phosphate on the surface of the copolymer, wherein the molar ratio of the hydroxyethyl methyl methacrylate monomer to the methyl methacrylate monomer is 1:3-1:15 (preferably 1: 4-1:12, more preferably 1:5-1:10).
  • HEMA methyl hydroxyethyl methacrylate
  • MMA methyl methacrylate
  • the copolymer has an average diameter d 1 in the range of 300 to 600 nm.
  • the bone material composite particles have an average diameter d 2 ranging from 400 to 900 nm, preferably from 500 to 800 nm, more preferably from 550 to 750 nm.
  • the d 2 :d 1 ranges from 1.05 to 1.2, preferably from 1.1 to 1.8, more preferably from 1.2 to 1.5.
  • the distribution of the bone material composite particles in the range of 200-900 nm particle size is ⁇ 95% (preferably ⁇ 96%).
  • the bone material composite particles have one or more characteristics selected from the group consisting of:
  • the bone graft formed by curing the bone material composite particles has a flexural strength of ⁇ 50 MPa (preferably 50-80 MPa);
  • the bone graft formed by curing the bone material composite particles has a compressive strength of ⁇ 100 MPa (preferably 100-150 MPa);
  • the operational curing time of the bone material composite particles ranges from 10 to 15 min, preferably from 12 to 15 min, more preferably from 14 to 15 min.
  • a method of preparing a bone material composite particle according to the first aspect of the present invention comprising the steps of:
  • HEMA methyl hydroxyethyl methacrylate
  • MMA methyl methacrylate
  • the composite particles are dried.
  • the step (1) is carried out at 60 to 100 ° C (preferably 65 to 95 ° C, more preferably 70 to 90 ° C, most preferably 75 to 85 ° C).
  • the step (1) comprises the step (1-1): in the potassium persulfate solvent, the hydroxyethyl methyl methacrylate monomer and the methyl methacrylate monomer are mixed and stirred.
  • the step (1) comprises: mixing the methyl ethyl methacrylate monomer and the methyl methacrylate monomer at 80 ° C under a nitrogen atmosphere, and stirring the reaction for 3 hours.
  • the mass ratio of the copolymer to Ca(OH) 2 is from 1:1 to 10:1, preferably from 2:1 to 8:1, more The good location is 3:1-7:1.
  • the drying is a lyophilization treatment.
  • a bone cement product comprising:
  • Component A comprising the composite particles according to the first aspect of the invention
  • Component B comprising methyl methacrylate and an accelerator selected from the group consisting of dimethyl-p-toluidine (DMPT), methyl ethyl ketone peroxide (MEKP), dicumyl peroxide , perester, decanoyl peroxide, tert-butane, tert-pentane, azobisisobutyronitrile (AIBN), neohexanoic acid, or a combination thereof.
  • DMPT dimethyl-p-toluidine
  • MEKP methyl ethyl ketone peroxide
  • AIBN azobisisobutyronitrile
  • the component A further comprises a catalyst and a developer.
  • the catalyst is selected from the group consisting of benzoyl peroxide, N,N-dimethylamino-4-benzyl laurate (DMAL, N, N-dimethylamino-4-benzyl laurate) , N,N-dimethylamino-4-benzyl oleate, or a combination thereof.
  • DMAL N,N-dimethylamino-4-benzyl laurate
  • DMAL N, N-dimethylamino-4-benzyl laurate
  • N,N-dimethylamino-4-benzyl oleate or a combination thereof.
  • the developer is selected from the group consisting of BaSO 4 , ZrO 2 , or a combination thereof.
  • the mass ratio of the composite particles to the catalyst ranges from 1:1 to 20:1, preferably from 2:1 to 18:1, more preferably 3:1-16:1, optimally 3:1-15:1.
  • the mass ratio of the composite particles to the developer ranges from 10:1 to 80:1, preferably from 15:1 to 70:1, more preferably It is 20:1-50:1.
  • the volume ratio of the methyl methacrylate to the accelerator is from 10:1 to 150:1, preferably from 15:1 to 20:1, more preferably.
  • the ground is 30:1-100:1.
  • the mass ratio of the component A to the component B is from 1:1 to 1:20, preferably from 1:2 to 1:15, more preferably from 1:3: 10.
  • component A is a solid and component B is a liquid.
  • the bone cement product has a static water contact angle in the range of 67-84°, preferably 70-80°.
  • the bone cement product has a static oil contact angle in the range of from 29 to 38 degrees, preferably from 32 to 37 degrees.
  • a method for non-therapeutic preparation of a bone graft in vitro comprising the steps of:
  • the curing is carried out in a mold.
  • a composite particle according to the first aspect of the invention or a bone cement product according to the third aspect of the invention for the preparation of a bone filler for treating a bone disease.
  • the bone disease is selected from the group consisting of osteonecrosis, osteoporosis, osteoarthritis, vertebroplasty, fracture, bone cyst, alveolar bone atrophy, subchondral bone defect, subchondral bone Cysts, vertebroplasty, maxillofacial surgery, plastic surgery, minimally invasive surgery.
  • Figure 1 shows the Fourier transform infrared spectrum of bone cement products 1, 2 and 3 and the Fourier transform infrared spectrum of commercially available PMMA bone cement materials, top and bottom for cement products 1, 2, 3 and PMMA bone cement material is sold.
  • Figure 2 shows the nuclear magnetic resonance spectrum of HEMA-MMA copolymer 1 ( Figure 2a), 2 ( Figure 2b) and 3 ( Figure 2c).
  • FIG 3 shows the thermogravimetric analysis of bone cement products 1, 2 and 3 and the thermogravimetric analysis of commercially available PMMA bone cement materials, wherein (a) bone cement product 1, (b) bone cement product 2, (c Bone cement product 3, (d) HEMA-MMA copolymer 2, (e) PMMA sample.
  • Figure 4 shows the SEM topography of the reference sample MMA (Figure 4a), HEMA-MMA copolymer 1 (Figure 4b), 2 (Figure 4c), 3 ( Figure 4d).
  • Figure 5 shows the SEM morphology of HEMA-MMA copolymer 1 attached calcium phosphate (Figure 5e), HEMA-MMA copolymer 2 attached calcium phosphate (Figure 5f), HEMA-MMA copolymer 3 attached calcium phosphate (Figure 5g) Figure.
  • Figure 6 shows a static water/oil contact angle test picture of MMA (reference sample), bone cement product 1, bone cement product 2, bone cement product 3 (top-down).
  • Figure 7 shows a HEMA/polymethyl methacrylate copolymer nano-calcium phosphate coating of transmission electron microscopy and selected electron diffraction images.
  • Figure 8 shows the results of EDX analysis of HEMA/polymethyl methacrylate copolymer nano-calcium phosphate coatings, where HEMA:MMA ratio: (a) 1:3, (b) 1:6, (c) 1:8 .
  • Figure 9 shows a force-displacement curve of bone cement extrusion measured using a 21G needle at a 15 mm/min extrusion speed.
  • Figure 10 shows CCK-8 cytotoxicity assays for bone cement products 1, 2, 3 and MMA materials using mouse fibroblasts (L929) and human osteoblast-like cells (MG63).
  • Figure 11 shows Live/Dead fluorescence staining of MG63 cells after 24 h, 48 h and 72 h co-culture.
  • Figure 12 shows Live/Dead fluorescent staining of L929 cells after 24 h, 48 h and 72 h co-culture.
  • Figure 13 shows the results of hemolysis test for bone cement products 1, 2, 3 and MMA materials.
  • Figure 14 shows SEM observation of cell attachment of commercially available MMA and bone cement product 2 (L929).
  • Figure 15 shows a red-calcium-positive reaction to bone cement products 2 and 3, PMMA (not coated with calcium phosphate), and staining with Alizarin Red S.
  • Figure 16 shows the analysis of rat muscle pathological staining data.
  • Figure 17 shows HE staining of rat heart, liver and kidney.
  • Figure 18 is a representation of a micro-CT scan of the left tibia of a guinea pig.
  • Figure 19 is a three-dimensional reconstruction model of the left tibia of a guinea pig scanned by Micro-CT.
  • Figure 20 is a diagram showing the staining of the left tibia of guinea pigs.
  • the present inventors have for the first time unexpectedly discovered a novel bone material replacement synthetic formulation, preparation method and use method.
  • the present invention simultaneously passes (1) preventing the non-adhesive dough stage; (2) nano-reforming the formulation component; (3) changing the formulation polymerization reaction to achieve an ultra-low viscosity to achieve a comparison of the fine needle cylinder (21G or smaller). Injection possibility.
  • the present invention has been completed on this basis.
  • the term “about” means that the value can vary by no more than 1% from the recited value.
  • the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the terms "containing” or “including” may be open, semi-closed, and closed. In other words, the terms also include “consisting essentially of,” or “consisting of.”
  • Calcium phosphate can generally be mixed in bone cement or as a surface treatment coating for implants.
  • a calcium phosphate layer including hydroxyapatite, has a composition similar to a natural bone material, and thus promotes the binding of bone cells and bone cement, promotes bone cell formation into new bone, and allows bone cement and bone to fuse with each other. This effect is also known as the bioactivity of bone cement.
  • a method is to add a biologically active substance to the bone cement, a part of which may be exposed to the solidified cement surface and provide biological activity.
  • Another alternative is to use your body fluids to create a natural bioactive layer on the surface of the bone cement.
  • the released material from the surrounding body fluid or bone cement may form deposits after the bone cement is implanted.
  • the composition of this deposit is generally a layer of calcium phosphate, which is a sign of biocompatibility, a process known as mineralization.
  • the bone material composite particles of the invention have excellent fluidity and handling property, and the operation curing time after the components are mixed ranges from 10 to 15 min (preferably 12 to 15 min, more preferably 14 to 15 min), and can be applied.
  • 21G syringes or syringes with larger inner diameters such as 20G, 19G, 18G, 17G, etc.).
  • the bone material composite granule and bone cement product of the present invention comprises a copolymer of mixed hydroxyethyl methyl methacrylate and methyl methacrylate having better biological activity, and physically coating a layer of phosphoric acid on the surface thereof.
  • Calcium which forms a polymer-based solid material in a controlled short period of time, has excellent in vivo stability and good mechanical strength after implantation.
  • the bone material composite particles and bone cement products of the invention have excellent fluidity and handling property, can achieve ultra-low viscosity, non-stick dough stage, have better rheological properties, and are favorable for using fine syringes
  • An injection for example, a needle injection (e.g., 21G) having a diameter of about 0.5 mm or less) is performed.
  • the bone material composite particles and the bone cement product of the present invention can be injected in a thicker syringe (for example, 18 G or more) at a later stage after the mixing of the components to suit the requirements of individual surgery.
  • the bone material composite particles and bone cement products of the present invention can be sterilized by radiation or gas without changing their properties.
  • the bone material composite particles and bone cement products of the present invention can be used for maxillofacial surgery, orthopedic surgery, spinal surgery, etc., such as osteonecrosis, osteoporosis, alveolar bone atrophy, subchondral bone of osteoarthritis, etc.
  • the condition can be used in vertebroplasty or maxillofacial surgery or plastic surgery.
  • Methyl methacrylate [Wako/139-02726]) (MMA, 98+%, containing 0.005% hydroquinone
  • Particle size was measured using a Zetasizer Nano dynamic light scattering. A small amount of the sample was placed in deionized water, and the sample solution was transparent, and the test was performed after ultrasonication for 2 hours.
  • thermogravimetric analyzer Q50, TA, USA.
  • Test method The instrument must be zeroed before loading. Weigh 3-10mg of pre-dried sample on platinum plate, increase from room temperature to 600 °C (the sample is completely decomposed), and the heating rate is 10 °C/min. The process was carried out under nitrogen protection with a sample carrier gas of 40 mL/min and an equilibrium carrier gas of 60 mL/min. The temperature at which the sample loses 10% by weight is defined as the thermal decomposition temperature of the material.
  • the morphology and morphology of the white powder before and after mechanical adhesion to CaP before uncured were observed by scanning electron microscopy (SEM).
  • the contact angle refers to the angle between the tangential line of the gas-liquid interface made at the intersection of gas, liquid and solid three phases through the boundary line between the liquid and the solid-liquid.
  • the solid surface is hydrophilic, that is, the liquid is relatively easy to wet the solid, and the smaller the angle, the better the wettability;
  • the solid surface is hydrophobic, that is, the liquid does not easily wet the solid.
  • the samples were subjected to static contact angle testing using a contact angle analyzer (SL600, Solon Information Technology Co, Shanghai, China). Under normal temperature and pressure conditions, the prepared test pieces were placed on the test platform, and the relevant parameters of the software settings were opened. 4 ⁇ l of deionized water and diiodomethane suspension were used to randomly take 7 positions on each test piece. The contact angle measurement is performed, the light intensity is observed and adjusted according to the video dynamic image, the data and the image are recorded, and the average value is taken as the static contact angle value of the material.
  • SL600 Solon Information Technology Co, Shanghai, China
  • Sample preparation Mixing according to the ratio in the curing reference table, spreading on a glass sheet in a thin paste form, and forming a rectangular sheet-like test piece.
  • HEMA-MMA copolymer 1 was obtained.
  • the HEMA-MMA copolymer 1 obtained in 1.1 was added to 450 ml of Ca(OH) 2 (concentration: 0.0085 g/cm 3 ) solution, stirring was continued, and 7.5 ml of H 3 PO 4 solution (concentration: 20%) was added, H 3 The dispersion rate of the PO 4 solution was 0.139 ml/hr, and stirring was continued until the solution was neutral.
  • the obtained crude product was centrifuged at 7,800 rpm for 15 minutes, washed, and the water in the nanoparticles was sublimed into a gaseous state by freeze-drying to dehydrate the material to obtain PMMA-HEMA/CaP powder I.
  • Example 1 The difference from 1.1 of Example 1 was that HEMA was 3.8 ml and MMA was 19.6 ml.
  • Steps 2.2 and 2.3 are the same as in Examples 1.2 and 1.3.
  • Example 1 The difference from 1.1 in Example 1 was that HEMA was 2.8 ml and MMA was 20.4 ml.
  • Steps 3.2 and 3.3 are the same as in Examples 1.2 and 1.3.
  • Example 1 The difference from 1.1 in Example 1 was that HEMA was 8.4 ml and MMA was 14.6 ml.
  • Steps C1.2 and C1.3 are the same as in Examples 1.2 and 1.3.
  • Example 2.1 In the same manner as in Example 2.1, only the HEMA-MMA copolymer C2 was prepared, and no calcium phosphate was coated.
  • 2991cm -1 is CH 3 stretching vibration peak
  • 2950cm -1 is CH 2 stretching vibration peak
  • 1436cm -1 is HCH deformation vibration peak
  • 1270 ⁇ 1100cm -1 is CO
  • the vibration peak, 700cm -1 is the HCH out-of-plane vibration. It can be concluded that there is no change in the infrared spectrum characteristic peaks of the four groups after adding CaP. It can be seen that the addition of calcium phosphate has no effect on the infrared spectrum peak of the bone cement product.
  • the absorption peak appearing at the position of 3480 cm -1 is the OH stretching vibration peak of the added modified monomer HEMA.
  • the appearance of this absorption peak indicates that the modified monomer participates in the polymerization reaction of the polymethyl methacrylate material, which proves the addition.
  • the HEMA monomer is polymerized with MMA.
  • FIG. 2 the nuclear magnetic resonance spectra of HEMA-MMA copolymer 1 (Fig. 2a), 2 (Fig. 2b) and 3 (Fig. 2c), respectively.
  • the chemical structure of the HEMA-MMA copolymer was characterized by 1 H NMR. It can be seen from Figures 2a-2c that all proton peaks on the HEMA-MMA copolymer can be resolved by nuclear magnetic resonance H-spectrum. Based on the characteristic proton peak integrated area ratio of the HEMA and MMA repeating units, the m/n value of the HEMA-MMA copolymer can be calculated, and the number average molecular weight of the HEMA-MMA copolymer can be calculated by the following formula.
  • M MMA represents the molecular weight of one MMA unit and M HEMA represents the molecular weight of a single HEMA unit.
  • Q represents the integrated area of the MMA unit characteristic peak (–COOC H 3 ⁇ , ⁇ 6.10 ppm), and P represents the integrated area of the HEMA unit characteristic peak (–C H 2 OH, ⁇ 3.68 ppm).
  • the number average molecular weight of the HEMA-MMA copolymer is shown in Table 1.
  • the modified bone cement As shown in the thermogravimetric analysis chart of Fig. 3, compared with the commercially available bone cement, the modified bone cement has no significant change in thermal stability, and (c) the thermal decomposition curve of the bone cement product 3 exhibits two decompositions.
  • the first decomposition step may be an unpolymerized MMA monomer.
  • the HEMA-MMA copolymer 2 of group (d) contains only C, H and O elements.
  • the adhesion of calcium phosphate in the modified bone cement can be compared from the figure, (b)>(c)>(a) .
  • HEMA-MMA copolymer 1 average particle size of 759.7nm
  • HEMA-MMA copolymer 3 has an average particle diameter of 585.2 nm
  • HEMA-MMA copolymer C2 has an average particle diameter of 180.1 nm.
  • the diameter of the copolymer to which CaP is attached is significantly larger than the HEMA-MMA precursor to which CaP is not attached.
  • Figure 6 shows static water/oil contact angle data for MMA (reference sample), bone cement product 1, bone cement product 2, bone cement product 3, as shown in Table 3.
  • the interplanar spacing of the crystal faces is calculated from the ring in the selected area electron diffraction table (Table 3), which conforms to the interplanar spacing of the standard hydroxyapatite.
  • Table 3 selected area electron diffraction table
  • the measured data were processed using SPSS (19.0) statistical software, t-test analysis (significant difference *p ⁇ 0.05, **p ⁇ 0.005, ***p ⁇ 0.0005)
  • Mouse fibroblasts (L929) and human osteoblast-like cells (MG63) were selected to study the cytocompatibility of bone cement materials, and cell viability was detected by CCK-8.
  • a cell suspension was prepared, with a cell count of about 10,000 cells/ml, about 200 ul per well, seeded into a 24-well plate, and 1 ml of culture medium was added.
  • the bone cement product 1, the bone cement product 2, the bone cement product 3, and the MMA were set as the experimental group, and four groups were set in parallel in each group, and the culture liquid control group and the blank control group were set at the same time.
  • the cells were placed in a 37 ° C incubator for about 6 hours, and a pre-UV-sterilized sample (about 3 * 3 mm in size) was added.
  • the cells were cultured in a 37 ° C incubator for 24 h/48 h/72 h, then 200 ul of CCK-8 working solution was added to each well, and the metabolic activity of the cells was measured by a microplate reader for 90 min, and the absorbance at 450 nm was measured.
  • Calcein-AM is used in combination with EthD-1 to simultaneously stain fluorescent cells and dead cells.
  • Staining step DPS was used to prepare a final concentration of 2 ⁇ M Calcein-AM, 4 ⁇ M EthD-1 staining solution; wash cells twice with DPBS to eliminate or dilute serum esterase activity (serum esterase can hydrolyze Calcein-AM to increase extracellular fluorescence intensity) Incubation; adding 150 ⁇ L of staining solution for 20 minutes at room temperature or 10-15 minutes at 37 ° C; photographing by fluorescence microscopy.
  • sample control 0.2 ml blood sample was added to the material incubation tube, and the vibration was further incubated for 60 min;
  • Negative control group The negative control (AN): 10 ml saline + 0.2 ml blood sample was incubated for 60 min. After centrifugation at 800 rpm for 5 min, the supernatant was taken and measured at 540 nm using an absorbance tester. Absorbance, calculate hemolysis rate (HR)
  • the hemolysis ratio (HR) of red blood cells can reflect the blood compatibility of the material.
  • the hemolysis rate indicates the amount of red blood cell disruption during contact of the sample with blood. The higher the hemolysis rate, the more broken red blood cells. Therefore, the lower the hemolysis rate value indicates the better the blood compatibility of the material.
  • the hemolysis rate of biomaterials meeting medical standards must be less than 5%. It can be seen that the hemolysis rate of each group of materials has reached the medical standard, and the bone cement product 2, that is, the HEMA-MMA (1:6)/CaP material has the best blood compatibility.
  • this experiment selected L929 cells to detect the cell adhesion of bone cement. After the cells were inoculated on the bone cement and cultured for 12 hours, the SEM results showed that the cells were well attached to the bone cement and the morphology was normal. Compared with the commercially available PMMA bone cement, the modified bone cement is more suitable for cell growth, indicating that it is good. The help of L929 forms a variety of three-dimensional structures to form tissue functions.
  • the cells cultured in the biomineralization reaction test form mineralized nodules in cells under conditioned medium.
  • the mineralization test can test the content of calcium salts, and the reddish calcium positive reaction is indicated by the Alizarin Red S staining method. After the cells were stained with Alizarin Red S, orange-red mineralized nodules appeared to show the biological activity of the material.
  • Each specimen was seeded into a Petri dish with 70,000 cells. After 14 days of culture, the culture solution was removed and the culture dish was washed with a buffer solution. 2.5% glutaraldehyde diluted with dimethylhydrazine buffer was fixed at room temperature for 60 minutes to remove the fixative solution. Rinse twice with deionized water. Add alcohol to dehydrate. Add 2% yarrow red (Alizarin Red S) for 2 minutes and rinse with deionized water. Repeat the above steps twice until the orange-red block appears and you can observe it with a microscope.
  • the calcium cement-containing bone cement formulation exhibited more orange-red areas, expressed a better mineralization reaction, and was more conducive to bone fusion.
  • the rats were first anesthetized with ether, and the rats were weighed and then anesthetized with 10% chloral hydrate by weight. After the rats were completely comatose, the rat hairs of the back muscles were removed and partially disinfected with iodine. Squeeze the iodine solution, cut and peel the skin by surgery, cut the incision about 8 mm in diameter at the back muscle, and bury the pre-sterilized bone cement product 2 (size about 5*5mm) into the muscle layer. The layers were carefully sutured to clean the residual blood on the body surface, and the cages were kept to prevent the rats from biting each other and cultured in a natural experimental environment. The rats were given penicillin through the abdominal cavity within 3-4 days after surgery to prevent inflammation of the wound.
  • the rats were euthanized; the muscle tissue and material parts of the back of the mouse in contact with the material were carefully cut out, cut into appropriate size with a scalpel, and then the heart, liver, spleen, kidney and lungs were taken out and then performed. Cutting.
  • the excised tissue was placed in a 50 ml centrifuge tube, fixed with Bouin's fixative for 24 hours, and the remaining tissues were fixed with 4% paraformaldehyde solution for 24 hours;
  • each tissue was soaked with distilled water, 50%, 70%, 80%, 90%, 95%, 100% (I), 100% (II) concentration of ethanol for gradient dehydration for 30 min each time;
  • Dipping wax three times of dipping wax, 40 minutes each time, then placing the tissues on the embedding rack for paraffin embedding;
  • HE staining Hematoxylin was added to the slides carrying the tissue sections to cover the tissue sections. After 15 minutes, the hematoxylin was removed, separated by dilute hydrochloric acid, and rinsed with distilled water. Then add the eosin solution for dyeing for 10 min;
  • Masson trichrome staining using iron hematoxylin staining working solution, staining for 15min, rinsing with distilled water, using differentiation solution for 2s, washing with water for 1min; bluening solution for bluening for 3s, after rinsing, red chrysanthemum red staining for 10min, water washing for 1min; Treatment with % phosphotungstic acid solution, the degree of staining was controlled by microscopic observation until the muscles were red, the collagen fibers were reddish, washed with water; the aniline blue liquid staining solution was counterstained (collagen fibers) for 1 min, washed with water, and then differentiated with 95% ethanol. 100% ethanol dehydrated transparent, neutral gum seal.
  • a tissue section was prepared and photographed using a fluorescent/phase contrast microscope model Eclipse TE 2000. Collagen fibers are blue, cytoplasm, muscle fibers and red blood cells are red, and the nucleus is blue.
  • the bone cement product 2 has a thin layer of collagen fibrous tissue at the implantation site.
  • the surface of the bone cement product 2 can form a layer of collagen, the surrounding tissue and cell growth of the material are normal, no pathological and allergic reactions, indicating that the material and tissue compatibility is good, and can be applied to the body.
  • Cardiomyocytes were normal, no edema, no hypertrophy, no necrosis or degeneration, no vacuoles or granules in the cytoplasm, no fibrosis in the myocardial interstitial, no inflammatory cell infiltration; liver HE staining as shown in Figure 17, each period The morphology of the liver cells was normal, no granules or vacuoles were formed, the cytoplasm was uniform, the hepatic lobules were clear and regular, the interstitial no hemorrhagic exudation, no inflammatory cell infiltration, no changes in fibrosis, hepatocyte necrosis, etc.; Compared with the kidney sections of the rats, the shape of normal glomeruli was observed in the sections of each period, no glassy changes and sclerosis, no thickening of the basement membrane, no inflammatory cell infiltration and necrosis. Epithelial cells have a clear structure, no deformation or congestion, no granules or vacuoles, no de
  • the bone cement product of the present invention has good tissue compatibility.
  • Bone cement product is injected into the guinea pig tibia subchondral bone
  • a guinea pig was used to perform a bone cement injection test of the tibia subchondral bone. Firstly, the guinea pigs were temporarily anesthetized with diethyl ether. The guinea pigs were weighed and then anesthetized with 10% chloral hydrate by weight. After the guinea pigs were completely comatose, the guinea pig hairs were removed, partially disinfected with iodine, and the iodine solution was wiped off with alcohol. Make an incision along the inside of the humerus to open the joint capsule of the knee joint to expose the cartilage tibia.
  • a drill bit with a depth of 5 mm was made on the inside of the tibial subchondral bone with a 1 mm drill bit.
  • Pre-sterilized bone cement product 2 or PMMA bone cement is mixed and injected into the drill hole, and carefully sealed with bone wax seal and layered to clean the surface residual blood, caged to prevent guinea pigs from biting each other, and in a natural experimental environment to cultivate. After 2 months of culture, the guinea pigs were euthanized and their left tibia was collected and stored in 10% formalin.
  • the German Bruker Sky Scan 1076 Micro-CT scanner was used to set the left tibia of the guinea pig at a setting of 3 mm pixel 18 mm, voltage 100 kV, exposure time 2356 ms, frame average 2, beam filter 1 mm aluminum, etc. The sample is scanned. After scanning, the original data is reconstructed in three dimensions using software.
  • the three-dimensional reconstruction showed significant new bone formation on the bone cement product 2, and the modified bone cement showed better new bone formation ability than the PMMA bone cement.
  • selected subchondral bone cyst area is of interest Area (ROI) and analysis of osteogenesis volume (BV/TV, %), trabecular bone separation (tb.sp, mm).
  • bone cement product 2 showed higher osteogenic volume and lower trabecular bone separation compared to PMMA bone cement.
  • the left tibia sample of the guinea pig was treated and the excess tissue was trimmed until the knee joint remained.
  • Dehydration The hard tissue was soaked with 50%, 70%, 90%, 100% ethanol, respectively, for gradient dehydration, and then infiltrated with toluene and methyl methacrylate.
  • Dipping wax and sectioning Embedding a mixture of methyl methacrylate, benzoyl peroxide and dimethyl p-methylaniline, storing in the dark at 4 ° C until the polymer block hardens, slicing the tissue, and finally the thickness It is between 40 ⁇ m and 70 ⁇ m.
  • a tissue section was prepared and photographed using a fluorescent/phase contrast microscope model Eclipse TE 2000.
  • bone cell layer was observed on the interface of the bone cement product 2, and the fiber layer was observed on the PMMA bone cement interface.
  • bone cement product 2 exhibits better bioactivity and osteoconductivity, resulting in a better new bone material interface that provides better mechanical support for subchondral bone cysts.

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Abstract

一种骨材料复合物颗粒、其制备方法和使用方法以及由该复合物颗粒制成的骨水泥。该骨材料复合物颗粒包括羟乙基甲基丙烯酸甲酯(HEMA)和甲基丙烯酸甲酯(MMA)的共聚物，以及，包覆在共聚物表面上的磷酸钙。合成的骨替代材料有更优越的生物相容性、骨传导性、流变性能、以及力学性能和机械性能。骨材料可用于骨坏死、骨质疏松、骨关节炎、椎体成形术、骨折、骨囊肿、牙槽骨萎缩、软骨下骨缺损、软骨下骨囊肿、椎体成形术、颌面外科、整形手术、微创手术等领域。

Description

新的骨材料替代合成配方、制备方法和使用方法 技术领域
本发明属于骨材料应用领域，具体地说，本发明涉及一种新的骨材料替代合成配方、制备方法和使用方法。
背景技术
聚甲基丙烯酸甲酯(PMMA)骨水泥作为临床骨组织修复材料已被广泛使用多年，尽管其具有较好的理化性能和机械性能，但其亲水性能较差、固化温度较高，导致骨水泥材料与骨组织之间相容性较差，难以与骨组织形成良好结合，并且骨水泥粘度较高，不利于针筒注射。另外，PMMA的固化温度过高(达到120℃)、与骨结合不充分、与骨组织间(特别是骨质疏松处)存在松动、错位甚至脱落现象，这些缺陷很容易导致术后各种并发症。植入目前的PMMA骨水泥后，骨水泥的表面会形成一层薄结缔组织，会损害植入物和骨之间的直接力传递，最终会导致假体的过早松动。
为了提高骨水泥与骨的相容性，一般可采用磷酸钙进行处理，可混合在骨水泥中，也可作为植入物的表面处理涂层。Miyazaki等人(J.Biomed.Mater.Res.2003,67A(4)1417-1423)研究在PMMA骨水泥加入MPS(甲基丙烯酰氧基丙基三甲氧基硅烷)和氯化钙以提高生物活性，结果表明，只有在加入非常高浓度的MPS和氯化钙到PMMA骨水泥中，水泥表面才会矿化。增加矿化添加剂也导致水泥的机械性能显著恶化。
美国专利US5264215B1公开了向骨水泥中加入4-MET单体(4-甲基丙烯酰氧基偏苯三酸酐)或4-META(4-甲基丙烯酰乙氧基苯三酸酐)和磷酸钙，特别是羟基磷灰石。这些骨水泥配方有未反应的单体添加剂可能对身体产生不良反应。
美国专利US8834845B2公开了一种生物活性骨水泥，植入后能够提供生物活性表面，并能促进磷酸钙在骨水泥的表面形成。
磷酸钙骨水泥(Calcium phosphate cements，CPCs)具有优良的生物相容性、生物活性和骨传导性。但是，无任何添加剂的CPCs通常由于液-固相分离导致注射性很差；此外，凝聚力较弱，往往在与血液或生物体液接触初期易瓦解；以及较差的机械特性，包括韧性、脆性和可靠性方面限制了其进一步的应用。
综上所述，本领域迫切需要研发出生物相容性好、生物结合性好、可塑性强、使用寿命长、机械性能优异的骨材料。
发明内容
本发明的目的在于提供一种新的骨材料替代合成配方、制备方法和使用方法。
本发明第一方面，提供一种骨材料复合物颗粒，所述复合物颗粒包括羟乙基甲基丙烯酸甲酯(HEMA)和甲基丙烯酸甲酯(MMA)的共聚物，以及，包覆在所述共聚物表面上的磷酸钙，其中，所述羟乙基甲基丙烯酸甲酯单体和甲基丙烯酸甲酯单体的摩尔比为1:3-1:15(较佳地为1:4-1:12，更佳地为1:5-1:10)。
在另一优选例中，所述共聚物的平均直径d1的范围为300-600nm。
在另一优选例中，所述骨材料复合物颗粒的平均直径d2范围为400-900nm，较佳地为500-800nm，更佳地为550-750nm。
在另一优选例中，所述d2：d1范围为1.05-1.2，较佳地为1.1-1.8，更佳地为1.2-1.5。
在另一优选例中，所述骨材料复合物颗粒在200-900nm粒径范围内的分布率≥95％(较佳地为≥96％)。
在另一优选例中，所述骨材料复合物颗粒具有选自下组的一个或多个特性：
(1)所述骨材料复合物颗粒固化形成的骨移植物的弯曲强度≥50MPa(较佳地为50-80MPa)；
(2)所述骨材料复合物颗粒固化形成的骨移植物的抗压强度≥100MPa(较佳地为100-150MPa)；和
(3)所述骨材料复合物颗粒的操作固化时间范围为10-15min，较佳地为12-15min，更佳地为14-15min。
本发明第二方面，提供一种制备如本发明第一方面所述骨材料复合物颗粒的方法，包括步骤：
(1)提供羟乙基甲基丙烯酸甲酯(HEMA)和甲基丙烯酸甲酯(MMA)的共聚物，其中，所述羟乙基甲基丙烯酸甲酯和甲基丙烯酸甲酯的摩尔比为1:3-1:15(较佳地为1:4-1:12，更佳地为1:5-1:10)；
(2)将所述共聚物、Ca(OH)2和H3PO4反应，制得表面包覆磷酸钙的共聚物，从而制得所述复合物颗粒；
(3)任选地，对复合物颗粒进行干燥。
在另一优选例中，所述步骤(1)在60-100℃(较佳地为65-95℃，更佳地为70-90℃，最佳地为75-85℃)下进行。
在另一优选例中，所述步骤(1)包括步骤(1-1)：过硫酸钾溶剂中，羟乙基甲基丙烯酸甲酯单体和甲基丙烯酸甲酯单体进行混合，搅拌。
在另一优选例中，所述步骤(1)包括：80℃，氮气保护下，羟乙基甲基丙烯酸甲酯单体和甲基丙烯酸甲酯单体进行混合，搅拌反应3小时。
在另一优选例中，所述步骤(2)中，所述共聚物和Ca(OH)2的质量比为1:1-10:1， 较佳地为2:1-8:1，更佳地为3:1-7:1。
在另一优选例中，所述步骤(3)中，所述干燥为冻干处理。
本发明第三方面，提供一种骨水泥产品，所述骨水泥产品包括：
(1)组分A，包括如本发明第一方面所述的复合物颗粒；和
(2)组分B，包括甲基丙烯酸甲酯和加速剂，所述加速剂选自下组：二甲基-对甲苯胺(DMPT)、过氧化甲乙酮(MEKP)、过氧化二异丙苯、过酸酯、过氧化癸酰、叔丁烷、叔戊烷、偶氮二异丁腈(AIBN)、新己酸、或其组合。
在另一优选例中，所述组分A还包括催化剂和显影剂。
在另一优选例中，所述催化剂选自下组：过氧化苯甲酰、N,N-二甲基氨基-4-十二酸苄酯(DMAL，N,N-dimethylamino-4-benzyl laurate)、N,N-二甲基氨基苄基油酸酯(DMAO，N,N-dimethylamino-4-benzyl oleate)、或其组合。
在另一优选例中，所述显影剂选自下组：BaSO4、ZrO2、或其组合。
在另一优选例中，所述组分A中，所述复合物颗粒和催化剂的质量比范围为1：1-20:1，较佳地为2:1-18:1，更佳地为3:1-16:1，最佳地为3:1-15:1。
在另一优选例中，所述组分A中，所述复合物颗粒和显影剂的质量比范围为10:1-80:1，较佳地为15:1-70：1，更佳地为20:1-50:1。
在另一优选例中，所述组分B中，所述甲基丙烯酸甲酯和加速剂的体积比为10:1-150:1，较佳地为15:1-120：1，更佳地为30:1-100:1。
在另一优选例中，所述组分A与组分B的质量比为1:1-1:20，较佳地为1:2-1:15，更佳地为1:3-1:10。
在另一优选例中，所述组分A为固体，且所述组分B为液体。
在另一优选例中，所述骨水泥产品的静态水接触角范围为67-84°，较佳地为70-80°。
在另一优选例中，所述骨水泥产品的静态油接触角范围为29-38°，较佳地为32-37°。
本发明第四方面，提供一种体外非治疗性地制备骨移植物的方法，包括步骤：
(a)提供本发明第一方面所述的骨材料复合物颗粒，
(b)将所述的骨材料复合物颗粒与固化液进行混合，形成混合物；和
(c)对所述混合物进行固化，形成所述骨移植物。
在另一优选例中，所述的固化是在模具进行。
本发明第五方面，提供一种如本发明第一方面所述的复合物颗粒或如本发明第三方面所述的骨水泥产品在制备治疗骨疾病的骨填充物中的应用。
在另一优选例中，所述骨疾病选自下组：骨坏死、骨质疏松、骨关节炎、椎体成形术、骨折、骨囊肿、牙槽骨萎缩、软骨下骨缺损、软骨下骨囊肿、椎体成形术、颌面外科、整形手术、微创骨手术。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了骨水泥产品1、2和3的傅里叶转换红外光谱以及市售PMMA骨水泥材料的傅里叶转换红外光谱，自上而下分别为骨水泥产品1、2、3以及市售PMMA骨水泥材料。
图2显示了HEMA-MMA共聚物1(图2a)、2(图2b)和3(图2c)的核磁共振波谱。
图3显示了骨水泥产品1、2和3的热重分析图以及市售PMMA骨水泥材料的热重分析图，其中，(a)骨水泥产品1、(b)骨水泥产品2、(c)骨水泥产品3、(d)HEMA-MMA共聚物2、(e)PMMA样品。
图4显示了参考样本MMA(图4a)、HEMA-MMA共聚物1(图4b)、2(图4c)、3(图4d)的SEM形貌图。
图5显示了HEMA-MMA共聚物1附着磷酸钙(图5e)、HEMA-MMA共聚物2附着磷酸钙((图5f)、HEMA-MMA共聚物3附着磷酸钙(图5g)的SEM形貌图。
图6显示了MMA(参考样本)、骨水泥产品1、骨水泥产品2、骨水泥产品3的静态水/油接触角测试图片(自上而下)。
图7显示了透射电镜和选区电子衍射图像的HEMA/聚甲基丙烯酸甲酯共聚物纳米磷酸钙涂层。
图8显示了HEMA/聚甲基丙烯酸甲酯共聚物纳米磷酸钙涂层的EDX分析结果，其中HEMA:MMA比例：(a)1:3，(b)1:6，(c)1:8。
图9显示了使用21G针在15毫米/分钟挤出速度下测定的骨水泥挤压的力-位移曲线图。
图10显示了采用小鼠成纤维细胞(L929)和人成骨样细胞(MG63)对骨水泥产品1、2、3和MMA材料进行CCK-8细胞毒性检测。
图11显示了MG63细胞共培养24h、48h和72h后的Live/Dead荧光染色。
图12显示了L929细胞共培养24h、48h和72h后的Live/Dead荧光染色。
图13显示了骨水泥产品1、2、3和MMA材料溶血率测试结果。
图14显示了市售MMA与骨水泥产品2(L929)细胞贴附SEM观察。
图15显示了对骨水泥产品2和3、PMMA(不包覆磷酸钙)、利用茜草红(Alizarin Red S)染色法示结节呈红色钙阳性反应。
图16显示了大鼠肌肉病理染色数据分析。
图17显示了大鼠心脏、肝脏和肾脏的HE染色。
图18为Micro-CT扫描豚鼠左胫骨代表图。
图19为Micro-CT扫描豚鼠左胫骨三维重建模型图。
图20为豚鼠左胫骨理染色图。
具体实施方式
本发明人通过广泛而深入的研究，首次意外地发现一种新型的骨材料替代合成配方、制备方法和使用方法。通过调整HEMA与MMA的比例、控制HEMA与MMA共聚物的磷酸钙包裹厚度和分布，提高骨水泥固化后的生物相容性以及矿化的效率。另外，本发明同时通过(1)防止无粘性面团阶段；(2)纳米化配方成份；(3)改变配方聚合反应，达到超低粘度，以达到比较幼细针筒(21G或较小)的注射可能性。在此基础上完成了本发明。
术语说明
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
磷酸钙
如本文所用，术语“磷酸钙”、“CaP”可互换使用。
磷酸钙一般可以混合在骨水泥中，也可以作为植入物的表面处理涂层。这样的磷酸钙层包括羟基磷灰石有一个类似天然骨材料的成份，并因此可以促进骨细胞和骨水泥的结合，促进骨细胞型成新的骨质，让骨水泥和骨相互融合。这种效应也被称为骨水泥的生物活性。为了对骨水泥提供生物活性，方法是在骨水泥加入生物活性物质，其中一部分磷酸钙可能暴露于已凝固的水泥面，并提供生物活性。另一种替代方法是利用自身的体液在骨水泥表面产生自然生物活性层。在植入骨水泥后来自周围体液或骨水泥的释放物质可能形成沉积物。这种沉积物的成份一般是磷酸钙层，是有利于生物相容性的表现，这个过程也被称为矿化。
流动性
本发明的骨材料复合物颗粒具有优异的流动性和操控性，组分混合后的操作固化时间范围为10-15min(较佳地为12-15min，更佳地为14-15min)，能够适用于21G针筒或内径更大的针筒(如20G、19G、18G、17G等)。
本发明的主要优点在于：
(1)本发明的骨材料复合物颗粒及骨水泥产品包含具有更好生物活性的混合羟乙基甲基丙烯酸甲酯与甲基丙烯酸甲酯的共聚物，并在其表面物理包裹一层磷酸钙，在可控制的短时间内形成聚合物基固体材料，体内稳定性优异，植入后机械强度良好。
(2)本发明的骨材料复合物颗粒及骨水泥产品具有优异的流动性和操控性，可达到超低粘度，无粘性面团阶段，具有更好的流变性能，有利于使用幼细针筒进行注射(例如直径约0.5mm或更小的针头注射(如21G))。并且，本发明的骨材料复合物颗粒及骨水泥产品可以在成份混合后的后阶段以较粗的针筒(例如18G或更大)进行注射，以适应个别手术的要求。
(3)本发明的骨材料复合物颗粒及骨水泥产品可以通过辐射或气体灭菌而不改变其性能。
(4)本发明的骨材料复合物颗粒及骨水泥产品可用于颌面外科、整形外科、脊柱外科等，例如骨坏死、骨质疏松、牙槽骨萎缩、骨性关节炎的软骨下骨等病症，可用于椎体成形术颌面外科手术或整形手术中。
原料
市售PMMA骨水泥：
Spineplex不透射线骨水泥(购自Stryker Corp)：
40g粉剂(4.7g聚甲基丙烯酸甲酯、含有过氧化苯甲酰(1.5％)的23.3g MMA-苯乙烯共聚物、12g硫酸钡)；以及
20ml液剂(19.5ml MMA、0.5ml N,N-二甲基-对甲苯胺、1.5mg对苯二酚)。
固化时间8.2min，10-14G针筒
MMA：
甲基丙烯酸甲酯([Wako/139-02726])(MMA,98+％，含0.005％对苯二酚
通用方法
1.傅立叶红外光谱(FTIR)分析
2.核磁共振波谱(NMR)分析
采用德国Bruker AV 400核磁共振仪(NMR)进行样品测试。选用四甲基硅烷(Tetramethylsilane TMS)为内标，用氘代二氯甲烷溶解样品。采用400MHz的频率常温下扫描32次得到核磁共振氢谱(1H NMR谱图)。
3.粒径分布分析
使用Zetasizer Nano动态光散射检测颗粒粒径。取少量样品置于分散于去离子水中，样品液呈透明状，超声2小时后进行测试。
4.TGA热重分析
使用热重分析仪(Q50，美国TA公司)检测材料热稳定性。
测试方法：装样之前必须对仪器进行调零，称取3-10mg预先干燥的样品置于铂金盘上，从室温升到600℃(样品完全分解)，升温速度为10℃/min，全过程在氮气保护下进行，样品载气40mL/min，平衡载气为60mL/min。以样品失重10wt％时的温度定为材料的热分解温度。
5.扫描电子显微镜(SEM)分析
使用电子扫描显微镜(SEM)观察未固化前、机械附着CaP前/后白色粉体，进行形貌、形态观察。
6、静态水接触角测试
接触角是指在气、液、固三相交点处所作的气-液界面的切线穿过液体与固-液交界线之间的夹角。
θ<90°，则固体表面是亲水性的，即液体较易润湿固体，其角越小，表示润湿性越好；
θ>90°，则固体表面是疏水性的，即液体不容易润湿固体。
使用接触角分析仪对样品进行静态接触角测试(SL600,Solon Information Technology Co,Shanghai,China)。在常温常压条件下，将事先制备的试件放于测试平台，打开软件设置相关参数，分别用4μl去离子水和二碘甲烷悬滴的方法，在每个试件上随机取7个位置进行接触角测量，根据视频动态图观察并调整光强，记录数据和图像，取平均值作为该种材料的静态接触角值。
试件制作：按照固化参照表中的比例混合，在稀糊状时铺展在玻璃片上，制成长方形薄片状试件。
7.透射电镜和选区电子衍射图像的HEMA/聚甲基丙烯酸甲酯共聚物纳米磷酸钙涂层
使用透射电子显微镜(TEM)和选区电子衍射图像观察磷酸钙涂层观察晶体样品的形貌特征与晶体学性质的原位分析
8.EDX分析的HEMA/聚甲基丙烯酸甲酯共聚物纳米磷酸钙涂层
使用搭配了能量色散X射线光谱的透射电子显微镜(TEM)进行元素分析
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1：骨水泥产品1
1.1乳液聚合法制备HEMA-MMA共聚物I(HEMA-MMA＝1：3(wt％))
80℃，氮气保护下，6.0ml HEMA(浓度95％)和17.2ml MMA(浓度98％)单体加入到120ml过硫酸钾溶液(浓度0.00133g/cm3)中，不断搅拌反应3小时，制得HEMA-MMA共聚物1。
1.2HEMA-MMA共聚物1上附着磷酸钙
将1.1中得到的HEMA-MMA共聚物1加入到450ml Ca(OH)2(浓度0.0085g/cm3)溶液中，不断搅拌，加入7.5ml的H3PO4溶液(浓度20％)，H3PO4溶液的分散率在0.139毫升/小时，不断搅拌直至溶液呈中性。将所得粗产物7800转/15分钟离心，洗涤，用冷冻干燥把纳米粒子中的水分升华成气态使物料脱水，制得PMMA-HEMA/CaP粉末I。
1.3HEMA-MMA/CaP骨水泥的固化
室温下，称取10g粗过氧化苯甲酰BPO溶于40ml氯仿中，滤去不溶物。滤液倒入l00ml预先用冰盐浴冷却的甲醇中，即有针状结晶析出，抽滤，在氯化钙存在下减压干燥，得精制品。
分别称取上述PMMA-HEMA/CaP粉末I 7.5g、BPO0.8g、BaSO40.16g(质量比约1:1:0.2)，粗研磨使之混合均匀，加入25ml的MMA和0.25ml的DMPT混合溶液(MMA:DMPT体积比1:0.01)，沿着同方向匀速搅拌约2分钟，用注射器缓缓吸取混合液，轻敲管壁赶去气泡，注射入预先准备的模具中。
实施例2：骨水泥产品2
2.1乳液聚合法制备HEMA-MMA共聚物2(HEMA-MMA＝1：6(wt％))
同实施例1的1.1，不同之处在于，HEMA为3.8ml，MMA为19.6ml。
步骤2.2和2.3同实施例1.2和1.3。
实施例3：骨水泥产品3
3.1乳液聚合法制备HEMA-MMA共聚物3(HEMA-MMA＝1：8(wt％))
同实施例1的1.1，不同之处在于，HEMA为2.8ml，MMA为20.4ml。
步骤3.2和3.3同实施例1.2和1.3。
对比例1：骨水泥产品C1
C1.1：乳液聚合法制备HEMA-MMA共聚物C1(HEMA-MMA＝1：2(wt％))
同实施例1的1.1，不同之处在于，HEMA为8.4ml，MMA为14.6ml。
步骤C1.2和C1.3同实施例1.2和1.3。
对比例2：HEMA-MMA共聚物C2
同实施例2.1，仅制备HEMA-MMA共聚物C2，不包覆磷酸钙。
结果分析
1.如图1所示，分别为骨水泥产品1、2和3的傅里叶转换红外光谱以及市售PMMA骨水泥材料的傅里叶转换红外光谱。四组光谱结果对比可以看出：
2991cm-1为CH3伸缩振动峰，2950cm-1为CH2伸缩振动峰，1750cm-1为C＝O双键伸缩振动峰，1436cm-1为H-C-H变形振动峰，1270～1100cm-1处为C-O振动峰，700cm-1处为H-C-H面外振动，由此可以得出加上CaP后四组红外光谱特征峰无变化，可以看出加入磷酸钙对骨水泥产品的红外光谱峰值没有影响。
3480cm-1位置处出现的吸收峰，为添加的改性单体HEMA中O-H伸缩振动峰，此吸收峰的出现说明改性单体参与了聚甲基丙烯酸甲酯材料的聚合反应，证明添加的HEMA单体与MMA发生了聚合反应。
2.如图2所示，分别为HEMA-MMA共聚物1(图2a)、2(图2b)和3(图2c)的核磁共振波谱。HEMA-MMA共聚物的化学结构通过1H NMR来进行表征，通过图2a-2c可以看到HEMA-MMA共聚物上所有的质子峰都可以通过核磁共振H图谱中反应出来。根据HEMA和MMA重复单元的特征质子峰积分面积比，可以计算出HEMA-MMA共聚物的m/n值，进而通过下面的公式计算出HEMA-MMA共聚物的数均分子量。
Figure PCTCN2017094133-appb-000001
其中，MMMA表示一个MMA单元的分子量，MHEMA表示单个HEMA单元的分子量。Q代表MMA单元特征峰(–COOCH 3–，δ6.10ppm)的积分面积，P代表HEMA单元特征峰(–CH 2OH，δ3.68ppm)的积分面积。HEMA-MMA共聚物数均分子量如表1所示。
表1 HEMA-MMA共聚物数均分子量
Figure PCTCN2017094133-appb-000002
3.如图3的热重分析图可以看出，对比市售骨水泥，改性后骨水泥在热稳定性方面并没有明显变化，(c)骨水泥产品3的热分解曲线呈现两个分解阶梯，第一个分解阶梯可能是未聚合的MMA单体。(d)组的HEMA-MMA共聚物2只含有C、H、O元素，从图中可对比出改性后骨水泥中磷酸钙的附着量，为(b)>(c)>(a)。
4.粒径分布结果：
(1)HEMA-MMA共聚物1平均粒径759.7nm；
(2)HEMA-MMA共聚物2平均粒径509.8nm；
(3)HEMA-MMA共聚物3平均粒径585.2nm；
(4)HEMA-MMA共聚物C2平均粒径180.1nm。
可以看出，附着CaP的共聚物直径明显大于未附着CaP的HEMA-MMA前体。
5.SEM分析
参考样本MMA(图4a)、HEMA-MMA共聚物1(图4b)、2(图4c)、3(图4d)，以及，骨水泥产品1(图5e)、2((图5f)、3(图5g)的SEM图如图4和图5所示。CaP包覆量及粒径大小如表2所示。
表2
编号 样品 CaP包覆量(g) 粒径(nm)
1 MMA(参考样本)   -
2 骨水泥产品C1 1-2 954±223
3 骨水泥产品1 1-2 629±139
4 骨水泥产品2 1-2 596±167
5 骨水泥产品3 1-2 405±103
6.静态水接触角测试结果
图6显示了MMA(参考样本)、骨水泥产品1、骨水泥产品2、骨水泥产品3的静态水/油接触角数据，具体如表3所示。
表3
Figure PCTCN2017094133-appb-000003
7.透射电镜和选区电子衍射(SAED)图像的HEMA/聚甲基丙烯酸甲酯共聚物纳米磷酸钙涂层
如图7所示，由选区电子衍射表(表3)中的环形计算晶面的晶面间距，其符合标准羟基磷灰石的晶面间距。因此，所述涂层中存在羟基磷灰石晶体。
表4
Figure PCTCN2017094133-appb-000004
8.使用21G针在15毫米/分钟挤出速度下测定骨水泥挤压的力-位移曲线，数据如表4所示。
表4
Figure PCTCN2017094133-appb-000005
9.CCK-8法细胞毒性检测
以亲水化改造后的不同配比丙烯酸骨水泥，通过CCK-8细胞毒性实验、溶血 实验等，比较它们对骨细胞的影响差异。
所测数据使用SPSS(19.0)统计软件处理，t检验分析(显著性差异*p<0.05，**p<0.005，***p<0.0005)
选择小鼠成纤维细胞(L929)和人成骨样细胞(MG63)来研究骨水泥材料的细胞相容性，细胞活力通过CCK-8检测。
实验步骤：制备细胞悬液，细胞计数约10000个/ml，每孔约200ul，接种到24孔板中，并加入1ml培养液。设置骨水泥产品1、骨水泥产品2、骨水泥产品3、MMA为实验组，每组平行设置四组，同时设置培养液控制组和空白对照组。
放置于37℃培养箱中培养约6小时，加入预先紫外灭菌消毒的试样(尺寸约3*3mm)。再放入37℃培养箱中分别培养24h/48h/72h，之后每孔加入200ul CCK-8工作液，培养90min用酶标仪测细胞的代谢活力，测定450nm的吸光度。
10.荧光染色
Calcein-AM与EthD-1结合使用，同时对活细胞和死细胞进行荧光染色。
染色步骤：用DPBS配置终浓度为2μM Calcein-AM，4μM EthD-1的染色液；用DPBS清洗细胞2次，消除或稀释血清酯酶活力(血清酯酶能够水解Calcein-AM增加细胞外荧光强度)；加入150μL染色液室温孵育20分钟或37℃温育10-15分钟；荧光显微镜观察拍照。如图11、12所示，根据LIVE/DEAD染色结果，MG63和L929细胞在24h各组材料细胞数目无明显差别，但细胞形态较为纤细，细胞未完全伸出伪足，说明材料对细胞造成一定影响；48h后各组材料的细胞形态与空白组相比基本一致，说明细胞状态良好，未明显观察到死细胞，但是细胞数目仍然较低，细胞增殖速度减慢，说明材料对细胞的增殖具有抑制作用；72h后细胞增殖速度大幅增加，细胞形态正常。
11.溶血率实验
取骨水泥产品1、2、3材料5*5mm，紫外消毒灭菌3小时；材料灭菌后用无菌超纯水洗涤3次，然后用PBS或者生理盐水洗涤3次；将材料转移至盛有10ml生理盐水的无菌试管中，37℃振动孵育60min。
准备血液样品：抗凝剂处理后的全血和生理盐水按4:5比例稀释；
实验组The sample control(AS)：取0.2ml血液样品加到材料孵育试管中，继续振动孵育60min；
阳性对照组The positive control(AP)：10ml超纯水+0.2ml血液样品振动孵育60min；
阴性对照组The negative control(AN):10ml生理盐水+0.2ml血液样品振动孵育60min；之后以800rpm离心5min，取上清液，使用吸光度测试仪在540nm测 量吸光度，计算溶血率(hemolysis rate,HR)
Figure PCTCN2017094133-appb-000006
如图13所示，红细胞的溶血率(hemolysis ratio,HR)能够反映材料的血液相容性。溶血率表示样品与血液接触过程中红细胞破碎的数量。溶血率数值越高，破损的红细胞越多。因此，越低的溶血率数值表示材料的血液相容性越好。满足医用标准的生物材料的溶血率数值必须低于5％。可以看各组材料的的溶血率均达到了医用标准，其中骨水泥产品2即HEMA-MMA(1:6)/CaP材料的血液相容性最优。
12.细胞贴附实验
将200ul高浓度细胞液滴于材料中心，在37℃恒温培养箱中培养6h，之后补加1.5ml培养液，没过材料，培养24小时，取出材料用PBS洗三次。放入预冷的固定液(2.5％戊二醛)3h，置于4℃冰箱。取出后用蒸馏水漂洗三次，置于冷冻干燥机干燥24小时以上。将样品用导电胶固定在工作台上，进行扫描电镜(SEM)观察。
如图14所示，本实验选择L929细胞检测骨水泥的细胞贴附性。细胞接种于骨水泥上并培养12h后，SEM结果显示细胞能够很好地贴附在骨水泥上，形态正常，对比市售PMMA骨水泥，改性后骨水泥更适合细胞生长，说明能够很好的帮助L929形成各种各样的立体结构来形成组织功能。
13.生物矿化反应试验
生物矿化反应试验所培养的细胞在条件培养基下细胞形成矿化结节的情况。矿化试验可试验钙盐类含量，利用茜草红(Alizarin Red S)染色法示结节呈红色钙阳性反应。细胞经茜草红(Alizarin Red S)染色后出现桔红色的矿化结节以显示材料的生物活性。每个标本以70,000细胞数种入培养皿中，培养14天后去除培养液以缓冲溶液清洗培养皿，使用二甲胂缓冲液稀释的2.5％戊二醛在在室温下固定60分钟，去除固定液再利用去离子水冲洗2次。再加入酒精脱水。加入2％茜草红(Alizarin Red S)放置2分钟，以去离子水冲洗。重复以上步骤2次，直到橘红色区块出现就可利用显微镜观察。
如图15所示，含有磷酸钙的骨水泥配方呈现出更多的橘红色区，表达了更好的矿化反应，更加有利于与骨融合。
14.大鼠背部肌肉病理染色分析：骨水泥产品注入大鼠背部肌肉
成年SD大鼠10只，体重在150-200g之间，在实验动物房饲养一周后，进行皮下肌肉包埋实验。
首先使用乙醚暂时麻醉大鼠，对大鼠称重后按重量使用10％的水合氯醛进行腹腔麻醉，等大鼠完全昏迷后，剔除背肌部位的鼠毛，用碘进行局部消毒，用酒 精擦去碘液，用手术剪切开并剥离皮肤，在背肌处割开直径大约8mm的切口，将预先消毒的骨水泥产品2(尺寸约5*5mm)试件埋入肌肉层，分层仔细缝合，清洁体表残留血液，分笼饲养防止大鼠互相撕咬，并在自然实验环境下进行培养。在术后的3-4天内对大鼠通过腹腔给青霉素，防止伤口发炎。
培养8周后，对大鼠进行安乐死；小心切取小鼠背部与材料接触的肌肉组织和材料部分，用手术刀切割成合适的大小形状，再依次取出心脏、肝脏、脾脏、肾脏和肺脏并进行切割。
将切取的组织放于50ml离心管中，用Bouin’s固定液固定24小时其余组织用4％的多聚甲醛溶液固定24小时；
脱水：分别用蒸馏水、50％、70％、80％、90％、95％、100％(I)、100％(II)浓度的乙醇浸泡各组织进行梯度脱水，每次30min；
透明：将各组织依次浸泡在乙醇和二甲苯1:1溶液、二甲苯(I)和二甲苯(II)中，各30min；
浸蜡：分三次浸蜡，每次40min，之后将各组织放在包埋架上进行石蜡包埋；
用手术刀修整包埋组织的石蜡块进行组织切片，用刷子将切下的蜡片连成一长条蜡带，在展片机上展平；选择较好的蜡片，放在载玻片上，用烘干机进行烘干；
梯度脱蜡：将载玻片依次浸入二甲苯(I)和二甲苯(II)(各10min)、100％(I)、100％(II)、90％(I)、90％(II)、80％、70％、50％乙醇溶液中(各5min)，蒸馏水洗进行脱蜡；
HE染色：在载有组织切片的载玻片上加入苏木素，使其覆盖组织切片，15min后将苏木精去除，使用稀盐酸进行分色，用蒸馏水漂洗。再滴加伊红溶液进行染色10min；
Masson三色法染色：使用铁苏木素染色工作液，染色15min，蒸馏水漂洗后使用分化液分化2s，水洗1min；蓝化液返蓝3s，漂洗后丽春红品红染色10min，水洗1min；之后1％磷钨酸液处理，通过显微镜观察控制染色程度，至肌肉呈红色，胶原纤维呈淡红色为止，水洗；苯胺蓝液染色液复染(胶原纤维)1min，水洗，之后用95％乙醇分化，100％乙醇脱水透明，中性树胶封片。
镜检：使用型号为Eclipse TE 2000的荧光/相差显微镜观察并拍照制作好的组织切片。胶原纤维呈蓝色，胞质、肌纤维和红细胞呈红色，胞核呈蓝色。
如图16所示，骨水泥产品2植入部位有一薄层胶原纤维组织。
材料植入10天后，有较多中性粒细胞和淋巴细胞浸润，材料与肌肉组织接触部位有疏松的显微组织生成，接触边缘胶原层有所增加，肌肉细胞与正常组织相比无变化。
植入20天后，材料周围仍有少量淋巴细胞浸润，材料与组织结合较紧密，有 致密纤维胶原层包裹在材料表面，植入部位肌肉组织生长良好，肌肉细胞形态正常均一。
植入30天后，植入部位无炎症细胞浸润，纤维组织没有继续增厚，肌肉组织生长正常。根据实验结果，骨水泥产品2材料表面能够形成一层胶原蛋白，材料周边组织和细胞生长正常，无病变和过敏反应，表明材料与组织相容性较好，可应用于体内。
15.大鼠心脏、肝脏和肾脏病理染色分析
注射过量麻醉剂处死实验动物，立刻取出心脏、肝脏和肾脏等器官，观察外表与正常器官无明显异常，颜色正常，没有出现出血坏死等现象。与正常大鼠的相比，各时段大鼠的心脏切片无明显异常。心肌细胞均正常，无水肿，无肥大变形，无坏死或变性，胞浆内无空泡或颗粒形成，心肌间质无纤维化，无炎症细胞浸润；肝脏HE染色如图17所示，各期的肝细胞形态排列正常，未见颗粒或空泡形成，细胞质均匀，肝小叶清晰规则，间质无出血渗出，无炎性细胞浸润，未见纤维化、肝细胞坏死等改变；与正常大鼠肾脏切片比较，各个时段的切片中可见正常的肾小球的形状，无玻璃样变和硬化，基底膜无增厚，未见炎性细胞浸润及坏死。上皮细胞结构清晰，无变形或充血，无颗粒或空泡形成，无变性及坏死。
根据不同时期重要脏器的HE病理染色，SD大鼠的心脏、肝脏、肾脏未见明显异常。表明本发明的骨水泥产品具有较好的组织相容性。
16.骨水泥产品注入豚鼠胫骨软骨下骨
使用豚鼠进行胫骨软骨下骨骨水泥注射实验。首先使用乙醚暂时麻醉豚鼠，对豚鼠称重后按重量使用10％的水合氯醛进行腹腔麻醉，等豚鼠完全昏迷后，剔除部位的豚鼠毛，用碘进行局部消毒，用酒精擦去碘液，沿髌骨的内侧制一个切口，打开膝关节关节囊，露出软骨胫骨。在胫骨软骨下骨的内侧用1毫米的钻头做有5毫米的深度一个钻口。将预先消毒的骨水泥产品2或PMMA骨水泥混合并且注入钻口，并用骨蜡密封及分层仔细缝合，清洁体表残留血液，分笼饲养防止豚鼠互相撕咬，并在自然实验环境下进行培养。培养2个月后，对豚鼠进行安乐死，并收集其左胫骨，保存在10％福尔马林。
如图18所示，采用德国Bruker Sky Scan 1076 Micro-CT扫描仪，在三维像素18毫米，电压100kV，曝光时间2356毫秒，帧平均2，束过滤1毫米铝等设定下，对豚鼠左胫骨样品进行扫描。扫描后利用软件对原始数据进行三维重建。
如图19所示，三维重建显示在骨水泥产品2上有明显新骨生成，对比PMMA骨水泥，改性后骨水泥表现出更好的新骨生成能力。另外选定软骨下骨囊肿区域为感兴趣 区域(ROI)，并针对成骨体积(BV/TV，％)、骨小梁分离(tb.sp，mm)进行分析。
如表5所示，骨水泥产品2对比PMMA骨水泥，显示出更高的成骨体积及更低的骨小梁分离。
表5
Figure PCTCN2017094133-appb-000007
硬组织切片和组织学染色
对豚鼠左胫骨样品进行处理，修剪多余组织，直至剩下膝关节。
脱水：分别用50％，70％，90％，100％浓度的乙醇浸泡硬组织进行梯度脱水，然后用甲苯和甲基丙烯酸甲酯进行渗透。
浸蜡及切片：利用甲基丙烯酸甲酯，过氧化苯甲酰和二甲基对甲基苯胺混合物进行包埋，在4℃黑暗中储存，直到聚合物块硬化，对组织进行切片，最终厚度在40μm至70μm之间。
姬姆萨染色
在1％甲酸中轻轻搅拌30秒，使样品暴露，水中快速漂洗；然后在姬姆萨染色液，4℃染色10分钟，水中快速漂洗多余染色液，经空气干燥。
镜检：使用型号为Eclipse TE 2000的荧光/相差显微镜观察并拍照制作好的组织切片。
如图20所示，在骨水泥产品2介面上观察到骨细胞层，而PMMA骨水泥介面上则观察到纤维层。相比之下骨水泥产品2表现出更佳的生物活性和骨传导性，形成更佳的新骨材料介面，提供更好的机械支撑软骨下骨囊肿。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 一种骨材料复合物颗粒，其特征在于，所述复合物颗粒包括羟乙基甲基丙烯酸甲酯(HEMA)和甲基丙烯酸甲酯(MMA)的共聚物，以及，包覆在所述共聚物表面上的磷酸钙，其中，所述羟乙基甲基丙烯酸甲酯单体和甲基丙烯酸甲酯单体的摩尔比为1:3-1:15。
  2. 如权利要求1所述的骨材料复合物颗粒，其特征在于，所述共聚物的平均直径d1的范围为300-600nm。
  3. 如权利要求1所述的骨材料复合物颗粒，其特征在于，所述骨材料复合物颗粒具有选自下组的一个或多个特性：
    (1)所述骨材料复合物颗粒的平均直径d2范围为400-900nm；
    (2)所述d2：d1范围为1.05-1.2；
    (3)所述骨材料复合物颗粒在200-900nm粒径范围内的分布率≥95％；
    (4)所述骨材料复合物颗粒固化形成的骨移植物的弯曲强度≥50MPa；
    (5)所述骨材料复合物颗粒固化形成的骨移植物的抗压强度≥100MPa；和
    (6)所述骨材料复合物颗粒的操作固化时间范围为10-15min。
  4. 一种制备如权利要求1所述骨材料复合物颗粒的方法，包括步骤：
    (1)提供羟乙基甲基丙烯酸甲酯(HEMA)和甲基丙烯酸甲酯(MMA)的共聚物，其中，所述羟乙基甲基丙烯酸甲酯和甲基丙烯酸甲酯的摩尔比为1:3-1:15(较佳地为1:4-1:12，更佳地为1:5-1:10)；
    (2)将所述共聚物、Ca(OH)2和H3PO4反应，制得表面包覆磷酸钙的共聚物，从而制得所述复合物颗粒；
    (3)任选地，对复合物颗粒进行干燥。
  5. 一种骨水泥产品，其特征在于，所述骨水泥产品包括：
    (1)组分A，包括如权利要求1所述的复合物颗粒；和
    (2)组分B，包括甲基丙烯酸甲酯和加速剂，所述加速剂选自下组：二甲基-对甲苯胺(DMPT)、过氧化甲乙酮(MEKP)、过氧化二异丙苯、过酸酯、过氧化癸酰、叔丁烷、叔戊烷、偶氮二异丁腈(AIBN)、新己酸、或其组合。
  6. 如权利要求5的骨水泥产品，其特征在于，所述组分A还包括催化剂和显影剂。
  7. 如权利要求5的骨水泥产品，其特征在于，所述催化剂选自下组：过氧化苯甲酰、N,N-二甲基氨基-4-十二酸苄酯(DMAL，N,N-dimethylamino-4-benzyl laurate)、N,N-二甲基氨基苄基油酸酯(DMAO，N,N-dimethylamino-4-benzyl oleate)、或其组合。
  8. 如权利要求5的骨水泥产品，其特征在于，所述组分B中，所述甲基丙烯酸甲酯和加速剂的体积比为10:1-150:1。
  9. 一种体外非治疗性地制备骨移植物的方法，其特征在于，包括步骤：
    (a)提供权利要求1所述的骨材料复合物颗粒，
    (b)将所述的骨材料复合物颗粒与固化液进行混合，形成混合物；和
    (c)对所述混合物进行固化，形成所述骨移植物。
  10. 一种如权利要求1所述的复合物颗粒或如权利要求5所述的骨水泥产品在制备治疗骨疾病的骨填充物中的应用。
  11. 如权利要求10所述的应用，其特征在于，所述骨疾病选自下组：骨坏死、骨质疏松、骨关节炎、椎体成形术、骨折、骨囊肿、牙槽骨萎缩、软骨下骨缺损、软骨下骨囊肿、椎体成形术、颌面外科、整形手术、微创骨手术。
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