WO2024239276A1 - 一种弱碱性的复合骨水泥材料及其制备方法和应用 - Google Patents

一种弱碱性的复合骨水泥材料及其制备方法和应用 Download PDF

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WO2024239276A1
WO2024239276A1 PCT/CN2023/096054 CN2023096054W WO2024239276A1 WO 2024239276 A1 WO2024239276 A1 WO 2024239276A1 CN 2023096054 W CN2023096054 W CN 2023096054W WO 2024239276 A1 WO2024239276 A1 WO 2024239276A1
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bone cement
bioactive glass
composite bone
cement material
weakly alkaline
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French (fr)
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潘浩波
崔旭
张�浩
李红龙
柳春玉
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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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
    • A61L27/02—Inorganic materials
    • A61L27/10—Ceramics or glasses
    • 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
    • A61L27/18—Macromolecular materials obtained otherwise than 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
    • 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
    • A61L27/58—Materials at least partially resorbable by the body

Definitions

  • the invention belongs to the technical field of medical materials and relates to a weakly alkaline composite bone cement material and a preparation method and application thereof.
  • PMMA bone cement is widely used in the field of orthopedic repair materials due to its high mechanical strength, good plasticity and rapid curing at room temperature. It is also gradually used in the field of covering and rapid vascularization of skin ulcers caused by diabetes.
  • PMMA Polymethyl methacrylate
  • it can only form a fiber wrapping after implantation in the body and cannot form a strong bone bonding interface with the bone. Therefore, after a period of time, the osteoporotic vertebral body that has been implanted is prone to slippage, displacement and other problems, which can cause back pain and adjacent vertebral fractures in patients, thus limiting the application of PMMA bone cement in the field of bone repair materials.
  • CN104922731A the invention name is composite bone cement precursor, strontium borate bioglass/polymethyl methacrylate composite bone cement preparation method and application
  • the bioactive glass can effectively give it bioactivity and bone integration performance after being introduced into the inert material matrix.
  • the composite bone cement disclosed in the patent will form a high alkaline microenvironment after degradation, which will bring certain safety hazards to the human body.
  • the excessively high alkaline microenvironment will inhibit the activity and functional expression of cells related to tissue regeneration, and even cause hemolysis; for example, when bioactive glass is used for skin repair, the high alkaline microenvironment formed after degradation will cause a burning sensation on the skin, which may increase local inflammation of the skin to a certain extent. Therefore, based on the high alkaline microenvironment formed after degradation after implantation in the body, the content of bioactive glass introduced into the matrix of the tissue repair composite material is often not high enough, which also limits the improvement of the biological properties of the composite material to a certain extent.
  • the present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art.
  • the present invention proposes a weakly alkaline composite bone cement material and its preparation method and application.
  • the composite bone cement material of the present invention includes weakly alkaline borosilicate bioactive glass and polymethyl methacrylate (PMMA).
  • PMMA polymethyl methacrylate
  • the weakly alkaline borosilicate bioactive glass is weakly alkaline after degradation. Therefore, in the composite bone cement material, the addition amount of weakly alkaline borosilicate bioactive glass is high, so that the composite bone cement material has high biological activity characteristics, and optimizes the in vivo and in vitro biological safety and tissue repair performance of the composite bone cement.
  • a first aspect of the present invention provides a weakly alkaline composite bone cement material.
  • a weakly alkaline composite bone cement material includes a solid component and a liquid component
  • the solid components include weakly alkaline borosilicate bioactive glass, polymethyl methacrylate (PMMA), and a developer;
  • the liquid component includes methyl methacrylate (MMA) and a polymerization activator;
  • the chemical composition of the weakly alkaline borosilicate bioactive glass is aXO ⁇ bB 2 O 3 ⁇ cP 2 O 5 ⁇ dSiO 2 ⁇ eY 2 O, wherein a, b, c, d and e are molar fractions, a is 4-39, b is 5-15, 17-35, 37-53 or 55-65, c is 1-10, d is 0-60, e is 2-12, X is one or more of Ca, Mg and Sr, and Y is Na and/or K.
  • the developer comprises zirconium oxide and/or barium sulfate.
  • the solid component further comprises benzoyl peroxide (BPO).
  • BPO benzoyl peroxide
  • the polymerization activator comprises N,N-dimethyl-p-toluidine and/or hydroquinone.
  • the value of e ranges from 5 to 10.
  • Y is both Na and K
  • e represents the sum of the molar fractions of Na 2 O and K 2 O.
  • the value of a ranges from 34 to 39.
  • X is Ca, Mg, and Sr at the same time
  • a represents the sum of the molar fractions of MgO, CaO, and SrO.
  • the chemical composition of the weakly alkaline borosilicate bioactive glass is 3Na2O ⁇ 2K2O ⁇ 8MgO ⁇ 25CaO ⁇ 27B2O3 ⁇ 2P2O5 ⁇ 27SiO2 ⁇ 6SrO , 3Na2O ⁇ 4K2O ⁇ 8MgO ⁇ 23CaO ⁇ 27B2O3 ⁇ 2P2O5 ⁇ 27SiO2 ⁇ 6SrO , 4Na2O ⁇ 6K2O ⁇ 8MgO ⁇ 20CaO ⁇ 27B2O3 ⁇ 2P2O5 ⁇ 27SiO2 ⁇ 6SrO or 3Na2O ⁇ 4K2O ⁇ 8MgO ⁇ 16CaO ⁇ 27B2O3 ⁇ 2P2O5 ⁇ 27SiO2 ⁇ 13SrO .
  • the weakly alkaline borosilicate bioactive glass also includes ZnO and/or CuO.
  • the number average molecular weight of the polymethyl methacrylate is 300,000-1.5 million; further preferably, the number average molecular weight of the polymethyl methacrylate is 400,000-1 million.
  • the mass percentage of the weakly alkaline borosilicate bioactive glass in the solid component is greater than 20% and less than or equal to 70%; further preferably, the mass percentage of the weakly alkaline borosilicate bioactive glass in the solid component is greater than 20% and less than or equal to 60%; more preferably, the mass percentage of the weakly alkaline borosilicate bioactive glass in the solid component is 30-50%.
  • the mass percentage of the developer in the solid component is 18-32%; further preferably, the mass percentage of the developer in the solid component is 20-30%.
  • the mass percentage of the benzoyl peroxide in the solid component is 0.1-1.6%; further preferably, the mass percentage of the benzoyl peroxide in the solid component is 0.2-1.5%.
  • the solid components in terms of mass percentage, include 20.1-60% weakly alkaline borosilicate bioactive glass, 30-61.9% polymethyl methacrylate (PMMA), and 10-39.9% developer.
  • the solid components in terms of mass percentage, include 20.1-60% weakly alkaline borosilicate bioactive glass, 8.5-59.7% polymethyl methacrylate (PMMA), 20-30% developer, and 0.2-1.5% benzoyl peroxide.
  • the mass percentage of methyl methacrylate (MMA) is 90-99.5%, preferably 95-99.5%.
  • the mass percentage of the polymerization activator is 0.5-10%, preferably 0.5-5%.
  • the content of hydroquinone does not exceed 50 ppm, preferably 30-50 ppm.
  • the liquid component comprises, by mass percentage, 95-99.5% of methyl methacrylate and 0.5-5% of N,N-dimethyl-p-toluidine.
  • the mass volume ratio of the solid component to the liquid component is (2.1-3.0) g:1 mL; further preferably, in the weakly alkaline composite bone cement material, the mass volume ratio of the solid component to the liquid component is (2.1-2.7) g:1 mL; more preferably, in the weakly alkaline composite bone cement material, the mass volume ratio of the solid component to the liquid component is (2.3-2.5) g:1 mL.
  • the solid component is in powder form.
  • the particle size of the weakly alkaline borosilicate bioactive glass is 30-55 ⁇ m, preferably 40-50 ⁇ m.
  • the particle size of the polymethyl methacrylate is 10-90 ⁇ m, preferably 20-80 ⁇ m.
  • the second aspect of the present invention provides a method for preparing a weakly alkaline composite bone cement material.
  • a method for preparing a weakly alkaline composite bone cement material comprises the following steps:
  • Preparation of solid components weighing raw material components for preparing the weakly alkaline borosilicate bioactive glass, mixing, melting, cooling, and crushing to obtain the weakly alkaline borosilicate bioactive glass, and then mixing the weakly alkaline borosilicate bioactive glass, polymethyl methacrylate, and a developer to obtain the solid component;
  • Preparation of the liquid component mixing the methyl methacrylate (MMA) and a polymerization activator to obtain the liquid component;
  • MMA methyl methacrylate
  • the solid components and liquid components are mixed.
  • the raw material components for preparing the weakly alkaline borosilicate bioactive glass include sodium salt, potassium salt, magnesium salt, calcium salt, strontium salt, silicon dioxide, boric acid, and phosphate.
  • the sodium salt, potassium salt, magnesium salt, calcium salt, strontium salt refers to the carbonate or bicarbonate corresponding to sodium, potassium, magnesium, calcium, and strontium.
  • the preparation process of the solid component further includes crushing and ball milling operations after cooling.
  • the solid component and the liquid component are mixed according to a certain proportion.
  • the third aspect of the present invention provides an application of a weakly alkaline composite bone cement material.
  • a tissue repair material comprises the composite bone cement material.
  • the tissue repair material comprises a skin repair material or a bone repair material.
  • the previous borosilicate bioactive glass has good biological activity, it cannot be incorporated into the PMMA bone cement matrix in large quantities.
  • One of the important reasons is that the degradation of the borosilicate bioactive glass will cause strong alkalinity, which is easy to cause biosafety problems, such as hemolysis and cytotoxicity.
  • the incorporation of a lower content of borosilicate bioactive glass does not significantly improve the biological effect of PMMA bone cement. Therefore, the present invention prepares weakly alkaline borosilicate bioactive glass by adjusting the components of the borosilicate bioactive glass, which significantly reduces the high alkaline pH effect caused by its immersion and degradation in a specific solution.
  • the composite bone cement material of the present invention includes weakly alkaline borosilicate bioactive glass.
  • the present invention adjusts the composition of the weakly alkaline borosilicate bioactive glass to reduce the pH value of its degradation product, thereby achieving the effect of weak alkalinity after degradation.
  • the weak alkalinity can greatly improve the survival rate of cells and significantly reduce the hemolytic reaction caused by high alkalinity, thereby significantly improving its in vitro cell compatibility.
  • osteogenesis requires a certain alkaline environment, only under relatively appropriate weak alkaline conditions can the directional osteogenic differentiation of stem cells be effectively mediated, osteoblast activity be enhanced, and osteoclast activity be inhibited; when the alkalinity is too high, it will inhibit the proliferation and differentiation of stem cells, inhibit the activity of osteoclasts, and damage the host tissue; therefore, in view of the theory of alkaline regulation of osteogenesis, the alkaline environment is not the higher the better, but requires a specific weak alkaline microenvironment.
  • the weak alkaline composite bone cement material provided by the present invention can provide the weak alkaline microenvironment required to exert the best osteogenesis-promoting effect.
  • the weakly alkaline borosilicate bioactive glass content in the composite bone cement material of the present invention accounts for more than 20% of the solid components, for example, 30-60%, which is significantly higher than the borosilicate bioactive glass content in the bone cement material in CN104922731A. Therefore, the present invention can significantly improve the biological properties of PMMA-based composite bone cement, such as bioactivity, bone integration and bone regeneration ability, in vivo and in vitro.
  • the preparation method of the composite bone cement material provided by the present invention has readily available raw materials, simple process, convenient operation and easy application.
  • Fig. 1 is the mechanical properties of composite bone cement materials of Examples 1-3;
  • FIG2 is a graph showing the hemolysis rates of the composite bone cement materials of Examples 1-3 and Comparative Examples 1-3;
  • FIG3 is a graph showing the hemolysis rate of the composite bone cement materials of Examples 4-6;
  • FIG4 shows the survival of hBMSCs cells in culture medium containing 10% (mass concentration) CCK-8 reagent at different pH values on days 1, 3 and 7;
  • FIG5 is a live cell staining image of hBMSCs cells under different pH conditions for 1, 3, 5 and 7 days;
  • FIG6 shows the ALP staining and ALPL gene mRNA expression levels of hBMSCs cells after osteogenic induction for 3, 5, 7 and 10 days under different pH conditions
  • Figure 7 shows (a) immunocytochemistry of COL1A1 after hMSCs cells were induced into osteogenic tissues under different pH conditions for 10 days; (b) relative fluorescence intensity; (c) mRNA expression level of COL1A1 gene; (d) Sirius red staining;
  • FIG8 shows (a) Alizarin red staining of hBMSCs cells after osteogenic induction at different pH values for 7, 10 and 14 days; (b) Alizarin red semi-quantification;
  • Figure 9 shows the osteogenic-related genes of hBMSCs cells after osteogenic induction for 3, 7, 10 and 14 days under different pH conditions SP7 mRNA expression level.
  • the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.
  • a composite bone cement material comprising a solid component and a liquid component
  • the solid components calculated by weight percentage, include 30% weak alkaline borosilicate bioactive glass, 60% polymethyl methacrylate (PMMA) powder, and 10% developer (ZrO 2 ).
  • the liquid component calculated by mass percentage, comprises 99.2% of methyl methacrylate (MMA) and 0.8% of N,N-dimethyl-p-toluidine (DMPT);
  • the chemical composition of weakly alkaline borosilicate bioactive glass is 3Na2O ⁇ 2K2O ⁇ 8MgO ⁇ 25CaO ⁇ 27B2O3 ⁇ 2P2O5 ⁇ 27SiO2 ⁇ 6SrO .
  • a method for preparing a weakly alkaline composite bone cement material comprises the following steps:
  • the weakly alkaline borosilicate bioactive glass accounts for 30% of the solid component by weight.
  • the weakly alkaline borosilicate bioactive glass, PMMA powder and ZrO2 are placed in a three-dimensional mixer and uniformly mixed (50 Hz, 2 hours) to obtain a solid component.
  • Methyl methacrylate (MMA) and N,N-dimethyl-p-toluidine (DMPT) are mixed to obtain a liquid component;
  • the solid component and the liquid component are mixed at a mass volume ratio of 2.4 g:1 mL and stirred evenly to obtain a composite bone cement material.
  • a method for preparing a weakly alkaline composite bone cement material wherein the weakly alkaline borosilicate bioactive glass accounts for 40% of the solid component by mass, polymethyl methacrylate (PMMA) powder accounts for 40%, and a developer (ZrO 2 ) accounts for 20%.
  • PMMA polymethyl methacrylate
  • ZrO 2 a developer
  • a method for preparing a weakly alkaline composite bone cement material wherein the weakly alkaline borosilicate bioactive glass accounts for 50% of the solid component by mass, polymethyl methacrylate (PMMA) powder accounts for 40%, and developer (ZrO 2 ) accounts for 10%.
  • PMMA polymethyl methacrylate
  • ZrO 2 developer
  • a weakly alkaline borosilicate bioactive glass was prepared, which was denoted as weakly alkaline borosilicate bioactive glass-1 and had a chemical composition of 3Na2O ⁇ 4K2O ⁇ 8MgO ⁇ 23CaO ⁇ 27B2O3 ⁇ 2P2O5 ⁇ 27SiO2 ⁇ 6SrO .
  • the preparation process is as follows: 5.23g Na 2 CO 3 , 27.08g K 2 CO 3 , 38.34g (MgCO 3 ) 4 ⁇ Mg(OH) 2 ⁇ 5H 2 O, 112.75g CaCO 3 , 169.7g H 3 BO 3 , 80.05g SiO 2: , 43.71g SrCO 3 , and 30.79g NaH 2 PO 4 ⁇ 2H 2 O are weighed into a container, and the remaining steps are completely consistent with the method for preparing weakly alkaline borosilicate bioactive glass described in Example 1.
  • the weakly alkaline borosilicate bioactive glass prepared in this example replaces the weakly alkaline borosilicate bioactive glass in Example 1 to prepare the composite bone cement material, and the remaining steps are completely consistent with Example 1.
  • a weakly alkaline borosilicate bioactive glass was prepared , which was denoted as weakly alkaline borosilicate bioactive glass-2 and had a chemical composition of 4Na2O ⁇ 6K2O ⁇ 8MgO ⁇ 20CaO ⁇ 27B2O3 ⁇ 2P2O5 ⁇ 27SiO2 ⁇ 6SrO .
  • the preparation process is as follows: 10.3g Na 2 CO 3 , 40.29g K 2 CO 3 , 38.34g (MgCO 3 ) 4 ⁇ Mg(OH) 2 ⁇ 5H 2 O, 97.26g CaCO 3 , 169.7g H 3 BO 3 , 80.05g SiO 2: , 43.71g SrCO 3 , and 30.79g NaH 2 PO 4 ⁇ 2H 2 O are weighed into a container, and the remaining steps are completely consistent with the method for preparing weakly alkaline borosilicate bioactive glass described in Example 1.
  • the weakly alkaline borosilicate bioactive glass prepared in this example replaces the weakly alkaline borosilicate bioactive glass in Example 1 to prepare the composite bone cement material, and the remaining steps are completely consistent with Example 1.
  • a weakly alkaline borosilicate bioactive glass was prepared , which was named weakly alkaline borosilicate bioactive glass-3 and had a chemical composition of 3Na2O ⁇ 4K2O ⁇ 8MgO ⁇ 16CaO ⁇ 27B2O3 ⁇ 2P2O5 ⁇ 27SiO2 ⁇ 13SrO .
  • the preparation process is as follows: 5.23g Na 2 CO 3 , 27.08g K 2 CO 3 , 38.34g (MgCO 3 ) 4 ⁇ Mg(OH) 2 ⁇ 5H 2 O, 75.96g CaCO 3 , 169.7g H 3 BO 3 , 80.05g SiO 2: , 91.03g SrCO 3 , and 30.79g NaH 2 PO 4 ⁇ 2H 2 O are weighed into a container, and the remaining steps are completely consistent with the method for preparing weakly alkaline borosilicate bioactive glass described in Example 1.
  • the weakly alkaline borosilicate bioactive glass prepared in this example replaces the weakly alkaline borosilicate bioactive glass in Example 1 to prepare the composite bone cement material, and the remaining steps are completely consistent with Example 1.
  • Example 1 Compared with Example 1, the only difference of Comparative Example 1 is that the composition of the borosilicate bioactive glass used is: 6Na2O ⁇ 8K2O ⁇ 8MgO ⁇ 16CaO ⁇ 27B2O3 ⁇ 2P2O5 ⁇ 27SiO2 ⁇ 6SrO (called conventional borosilicate bioactive glass), and the rest of the process is the same as Example 1.
  • the composition of the borosilicate bioactive glass used is: 6Na2O ⁇ 8K2O ⁇ 8MgO ⁇ 16CaO ⁇ 27B2O3 ⁇ 2P2O5 ⁇ 27SiO2 ⁇ 6SrO (called conventional borosilicate bioactive glass), and the rest of the process is the same as Example 1.
  • Example 2 Compared with Example 2, the only difference of Comparative Example 2 is that the composition of the borosilicate bioactive glass used is: 6Na2O ⁇ 8K2O ⁇ 8MgO ⁇ 16CaO ⁇ 27B2O3 ⁇ 2P2O5 ⁇ 27SiO2 ⁇ 6SrO (called conventional borosilicate bioactive glass), and the rest of the process is the same as Example 2.
  • the composition of the borosilicate bioactive glass used is: 6Na2O ⁇ 8K2O ⁇ 8MgO ⁇ 16CaO ⁇ 27B2O3 ⁇ 2P2O5 ⁇ 27SiO2 ⁇ 6SrO (called conventional borosilicate bioactive glass), and the rest of the process is the same as Example 2.
  • Example 3 Compared with Example 3 , the only difference of Comparative Example 3 is that the composition of the borosilicate bioactive glass used is: 6Na2O ⁇ 8K2O ⁇ 8MgO ⁇ 16CaO ⁇ 27B2O3 ⁇ 2P2O5 ⁇ 27SiO2 ⁇ 6SrO (called conventional borosilicate bioactive glass), and the rest of the process is the same as Example 3.
  • the composition of the borosilicate bioactive glass used is: 6Na2O ⁇ 8K2O ⁇ 8MgO ⁇ 16CaO ⁇ 27B2O3 ⁇ 2P2O5 ⁇ 27SiO2 ⁇ 6SrO (called conventional borosilicate bioactive glass), and the rest of the process is the same as Example 3.
  • the composite bone cement material of Example 1-3 was injected into a mold, cured at room temperature for 1 hour, and then taken out to obtain a composite bone cement specimen with a length of (75 ⁇ 0.1) mm, a width of (10 ⁇ 0.1) mm, and a thickness of (3.3 ⁇ 0.1) mm, and four-point bending strength and bending modulus tests were performed.
  • the composite bone cement material of Example 1-3 was injected into the mold, cured at room temperature for 1 hour, and then taken out to obtain a cylindrical specimen, and the size after grinding reached a composite bone cement material with a length of (12 ⁇ 0.1) mm and a diameter of (6 ⁇ 0.1) mm. Material samples are tested for compressive strength.
  • Figure 1 shows the mechanical properties of the composite bone cement materials of Examples 1-3;
  • the horizontal axis "weakly alkaline borosilicate bioactive glass content" in Figure 1 indicates that the mass percentage of weakly alkaline borosilicate bioactive glass in the solid component of the composite bone cement material is 30%, 40%, and 50%, respectively, corresponding to Example 1, Example 2, and Example 3, respectively;
  • the dotted line in Figure 1 is the standard value specified in YY 0459-2003) is the four-point bending strength test result
  • (b) in Figure 1 is the flexural modulus test result
  • (c) in Figure 1 is the compressive strength test result.
  • the four-point bending strengths measured for the composite bone cement material specimens of Examples 1-3 are 54.81 ⁇ 1.73MPa (Example 1), 54.14 ⁇ 1.93MPa (Example 2) and 53.29 ⁇ 2.01MPa (Example 3), respectively;
  • the flexural modulus is 3766.02 ⁇ 83.93MPa (Example 1), 3861.51 ⁇ 74.19MPa (Example 2) and 3866.86 ⁇ 62.79MPa (Example 3), respectively;
  • the compressive strength is 86.28 ⁇ 3.18MPa (Example 1), 90.64 ⁇ 2.96MPa (Example 2) and 88.71 ⁇ 3.63MPa (Example 3).
  • the composite bone cement material samples prepared by Examples 1-3 were immersed in physiological saline and incubated in a 37° C. incubator for 2 hours. The pH value of the immersion solution was measured. The same experiment was performed on Comparative Examples 1, 2, and 3.
  • the pH values of the immersion solutions of the composite bone cement materials of Examples 1-3 were 8.89 (Example 1), 8.93 (Example 2) and 9.12 (Example 3), respectively, while the pH values of the conventional borosilicate bioactive glass composite bone cement materials were 10.46 (Comparative Example 1), 10.73 (Comparative Example 2) and 10.81 (Comparative Example 3), indicating that the alkaline pH of the composite bone cement material samples of Examples 1-3 after degradation was significantly reduced, thereby optimizing their biosafety in the body.
  • FIG. 2 shows the hemolysis rates of the composite bone cement materials of Examples 1-3 and the composite bone cement materials of Comparative Examples 1-3.
  • the hemolysis rates of the composite bone cement materials of Examples 1-3 are 1.39 ⁇ 0.21% (Example 1), 2.34 ⁇ 0.35% (Example 2), and 4.69 ⁇ 0.29% (Example 3), respectively, and the corresponding hemolysis rates of Comparative Examples 1-3 are 23.47 ⁇ 1.24% (Comparative Example 1), 42.06 ⁇ 2.25% (Comparative Example 2), and 49.18 ⁇ 1.99% (Comparative Example 3), respectively, indicating that the composite bone cement materials prepared in Examples 1-3 have a much lower hemolysis rate than the composite bone cement materials of Comparative Examples 1-3, and have better biosafety.
  • hBMSCs human bone marrow mesenchymal stem cells
  • hBMSCs cells were seeded at 5000 cells/well in a 96-well plate and cultured in ⁇ -MEM culture medium with different pH values for 1, 3 and 7 days, respectively.
  • the culture medium was aspirated and replaced with a culture medium containing 10% (mass concentration) CCK-8 reagent (serum-free) and incubated in a cell culture incubator for 1 hour.
  • the absorbance was detected at 450 nm using a microplate reader. The results are shown in FIG4 .
  • Figure 4 shows the survival status of hBMSCs cells on days 1, 3 and 7 in culture medium containing 10% (mass concentration) CCK-8 reagent at different pH values
  • Control in Figure 4 indicates the control group
  • Absorbance@450nm indicates the absorbance corresponding to 450nm
  • Time (day) indicates “time (day)”
  • * indicates p ⁇ 0.05
  • ** indicates p ⁇ 0.01
  • *** indicates p ⁇ 0.001
  • **** indicates p ⁇ 0.0001 compared with the Control group).
  • hBMSCs cells can maintain normal physiological metabolic activities within the pH range of 7.350-7.800. This shows that a weakly alkaline environment is essential for the physiological condition of cells.
  • hBMSCs cells were seeded at 4*10000/well in a 24-well plate and cultured in ⁇ -MEM culture medium with different pH values for 1, 3, 5 and 7 days, respectively.
  • the culture medium was then aspirated and Calcein-AM/PI reagent (live cell and dead cell double staining reagent) was added and incubated in a cell culture incubator for 30 minutes.
  • the live and dead states of the cells were observed using a fluorescence microscope. The results are shown in FIG5 .
  • Figure 5 shows live cell staining images of hBMSCs cells under different pH conditions for 1, 3, 5 and 7 days
  • Control represents the control group
  • Day1", “Day3”, “Day5" and “Day7” represent day 1, day 3, day 5 and day 7 respectively.
  • Figure 6 shows the ALP staining and mRNA expression level of ALPL gene of hBMSCs cells after osteogenic induction for 3, 5, 7 and 10 days under different pH conditions
  • Control represents the control group
  • Day3 represents the control group
  • Day5 represents the 3rd day, 5th day, 7th day and 10th day respectively
  • Day7 represents the 3rd day, 5th day, 7th day and 10th day respectively
  • Day10 represents the 3rd day, 5th day, 7th day and 10th day respectively
  • “Fold change” represents the fold difference
  • “Time (day)” represents time (day)).
  • Figure 6 (a) shows the results of ALP staining of hBMSCs cells.
  • the blue-purple part is ALP, and the darker the color, the higher the enzyme content.
  • the color of ALP staining in each group deepens, indicating that the expression of ALP increases.
  • the color of the pH 7.650 group is darker than that of the control group, and similarly at 7 days, it is also significantly darker than the control group.
  • the color of the pH 7.850 group is not significantly different from that of the control group.
  • the mRNA expression level of the ALPL gene was detected by qPCR (as shown in (b) in Figure 6).
  • Figure 7 (a) and (b) show the cell immunofluorescence images and semi-quantitative analysis of relative fluorescence intensity of hBMSCs cells at 10 days of osteogenic induction.
  • the green part is collagen type I alpha 1 chain (COL1A1) protein.
  • COL1A1 protein As can be seen from the figure, under the condition of acidity (pH 7.050) compared with the physiological environment, the expression of COL1A1 protein is significantly lower than that of the control group, with a significant difference. However, the expression is higher in the pH range of 7.650-7.800, and there is a significant difference between the pH 7.650 group and the control group.
  • the collagen of hBMSCs cells was further stained with Sirius red dye, and the red part is collagen.
  • the results are shown in Figure 7 (d).
  • the expression of collagen decreased, and it was found that the morphology and proliferation of the cells changed significantly.
  • the proliferation of cells was inhibited, and the cell morphology shrank.
  • the expression of collagen increased.
  • the expression of type I collagen did not increase with the increase of alkalinity.
  • the collagen expression of the pH 7.950 group decreased significantly, and it was found that the cell morphology also changed significantly.
  • hBMSCs cells only promoted collagen expression under conditions that were slightly alkaline (7.650-7.800) compared to physiological conditions. pH conditions beyond a certain range affected cell survival and cell morphology, which in turn affected cell protein expression under these conditions. This was consistent with the results of the expression level of the mRNA of the COL1A1 gene. Combined with the results of cell immunofluorescence, mRNA expression level, and Sirius red staining of COL1A1, it was shown that an alkaline environment within a certain range (pH 7.650-7.800) could promote the expression of type I collagen in hMSCs cells, while an environment beyond this range may affect cell metabolism, thereby reducing protein expression.
  • Figure 8 shows (a) Alizarin red staining of hBMSCs cells after osteogenic induction under different pH conditions for 7, 10 and 14 days; (b) Alizarin red semi-quantification (in Figure 8, "Day” means day, “Alizarin red S” means Alizarin red staining, and “control” means control group; “Alizarin red S Absorbance@562nm” means the absorbance of Alizarin red staining at 562nm, and "Time(day)” means time (day); “*” means p ⁇ 0.05, “**” means p ⁇ 0.01, "***” means p ⁇ 0.001, and “****” means p ⁇ 0.0001 compared with the control group).
  • the expression of the osteogenic-related gene SP7 was detected at 3, 7, 10 and 14 days after osteogenic induction, and the results are shown in FIG9 .
  • FIG9 shows the mRNA expression level of the osteogenic-related gene SP7 in hBMSCs cells after osteogenic induction for 3, 7, 10 and 14 days under different pH conditions (“Flod change” indicates the fold change, and “Time (day)” indicates time (day); “*” indicates p ⁇ 0.05, “**” indicates p ⁇ 0.01).
  • the expression of the SP7 gene increased with the increase of the number of days, and it was found that the expression level of the SP7 gene in the pH 7.650 group was significantly upregulated compared with the control group.

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Abstract

本发明属于医用材料技术领域，公开了一种弱碱性的复合骨水泥材料及其制备方法和应用。该复合骨水泥材料包括固体组分和液体组分；固体组分包括弱碱性硼硅酸盐生物活性玻璃，弱碱性硼硅酸盐生物活性玻璃的化学组成为aXO·bB2O3·cP2O5·dSiO2·eY2O，其中a、b、c、d和e为摩尔份数，a为4-39，b为5-15、17-35、37-53或55-65，c为1-10，d为0-60，e为2-12，X为Ca、Mg、Sr中的一种或多种，Y为Na或K中的一种或多种。本发明通过调整弱碱性硼硅酸盐生物活性玻璃的组成，达到降解后弱碱性的效果，显著降低高碱性导致的溶血反应，从而显著地提高了其体外细胞相容性。

Description

一种弱碱性的复合骨水泥材料及其制备方法和应用 技术领域
本发明属于医用材料技术领域，涉及一种弱碱性的复合骨水泥材料及其制备方法和应用。
背景技术
随着老龄化社会的加剧，基于老龄化导致的组织修复再生需求已成为社会面临的主要问题之一，比如骨质疏松性骨折直接导致了骨修复生物材料需求量的极大提高。
聚甲基丙烯酸甲酯(PMMA)骨水泥因其具力学强度高，有良好的可塑性及室温下快速固化等优点，被广泛用于骨科修复材料技术领域，也逐渐应用于糖尿病导致的皮肤溃疡的覆盖和快速血管化领域。但是由于PMMA的生物惰性，植入体内后只能形成纤维包裹，无法与骨形成牢固的骨性结合界面。因此植入缺损的骨质疏松椎体一段时间后，易发生滑脱、移位等问题，重新并发性引起患者背部疼痛和邻近椎体骨折，从而限制了PMMA骨水泥在骨修复材料领域中的应用。
针对以上缺点，已有研究团队将生物活性玻璃掺入PMMA骨水泥基体中从而赋予PMMA骨水泥一定的生物活性和骨整合性，如CN104922731A(发明名称为复合骨水泥前体、锶硼酸盐生物玻璃/聚甲基丙烯酸甲酯复合骨水泥的制备方法和应用)，该专利公开了生物活性玻璃降引入惰性材料基体后，能有效地赋予其生物活性以及骨整合性能。但是该专利公开的复合骨水泥降解后会形成较高碱性微环境，对人体会带来一定的安全隐患。比如生物活性玻璃基材料植入局部骨组织后，过高的碱性微环境会抑制组织再生相关细胞的活性和功能表达，甚至导致溶血；又如生物活性玻璃用于皮肤修复，其降解后形成的较高碱性微环境会引起皮肤灼烧感，从而一定程度上可能增加皮肤局部炎症。因此，基于植入体内降解后形成的较高碱性微环境，组织修复复合材料基体中引入生物活性玻璃的含量往往不够高，从而也一定程度上限制对于复合材料生物学性能的提升。
因此，需要优化生物活性玻璃降解后形成的过高碱性微环境，以实现复合基体的高含量生物活性玻璃掺杂，从而制备兼具高生物活性和低碱性的复合材料，以实现更优的体内生物安全性和组织再生修复性能。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此，本发明提出一种弱碱性的复合骨水泥材料及其制备方法和应用。本发明所述复合骨水泥材料中包括弱碱性硼硅酸盐生物活性玻璃、聚甲基丙烯酸甲酯(PMMA)，弱碱性硼硅酸盐生物活性玻璃降解后呈弱碱性，因此，在复合骨水泥材料中，弱碱性硼硅酸盐生物活性玻璃添加量高，使得所述复合骨水泥材料具有高的生物活性特点，优化了复合骨水泥的体内外生物安全性和组织修复性能。
本发明的第一方面提供一种弱碱性的复合骨水泥材料。
具体的，一种弱碱性的复合骨水泥材料，包括固体组分和液体组分；
所述固体组分包括弱碱性硼硅酸盐生物活性玻璃、聚甲基丙烯酸甲酯(PMMA)、显影剂；
所述液体组分包括甲基丙烯酸甲酯(MMA)和聚合活化剂；
所述弱碱性硼硅酸盐生物活性玻璃的化学组成为aXO·bB2O3·cP2O5·dSiO2·eY2O，其中a、b、c、d和e为摩尔份数，a为4-39，b为5-15、17-35、37-53或55-65，c为1-10，d为0-60，e为2-12，X为Ca、Mg、Sr中的一种或多种，Y为Na和/或K。
优选的，所述显影剂包括氧化锆和/或硫酸钡。
优选的，所述固体组分还包括过氧化苯甲酰(BPO)。
优选的，所述聚合活化剂包括N,N-二甲基对甲苯胺和/或对苯二酚。
优选的，所述弱碱性硼硅酸盐生物活性玻璃中，e的取值范围为5-10。当Y同时为Na和K时，e表示Na2O和K2O的摩尔份数之和。
优选的，所述弱碱性硼硅酸盐生物活性玻璃中，a的取值范围为34-39。当X同时为Ca、Mg、Sr时，a表示MgO、CaO和SrO的摩尔份数之和。
优选的，所述弱碱性硼硅酸盐生物活性玻璃的化学组成为3Na2O·2K2O·8MgO·25CaO·27B2O3·2P2O5·27SiO2·6SrO、3Na2O·4K2O·8MgO·23CaO·27B2O3·2P2O5·27SiO2·6SrO、4Na2O·6K2O·8MgO·20CaO·27B2O3·2P2O5·27SiO2·6SrO或3Na2O·4K2O·8MgO·16CaO·27B2O3·2P2O5·27SiO2·13SrO。
优选的，所述弱碱性硼硅酸盐生物活性玻璃中还包括ZnO和/或CuO。
优选的，所述聚甲基丙烯酸甲酯的数均分子量为30万-150万；进一步优选的，所述聚甲基丙烯酸甲酯的数均分子量为40万-100万。
优选的，所述弱碱性硼硅酸盐生物活性玻璃在所述固体组分中的质量百分含量为大于20％，小于等于70％；进一步优选的，所述弱碱性硼硅酸盐生物活性玻璃在所述固体组分中的质量百分含量为大于20％，小于等于60％；更优选的，所述弱碱性硼硅酸盐生物活性玻璃在所述固体组分中的质量百分含量为30-50％。
优选的，所述显影剂在所述固体组分中的质量百分含量为18-32％；进一步优选的，所述显影剂在所述固体组分中的质量百分含量为20-30％。
优选的，所述过氧化苯甲酰在所述固体组分中的质量百分含量为0.1-1.6％；进一步优选的，所述过氧化苯甲酰在所述固体组分中的质量百分含量为0.2-1.5％。
优选的，所述固体组分，按照质量百分含量计，包括弱碱性硼硅酸盐生物活性玻璃20.1-60％、聚甲基丙烯酸甲酯(PMMA)30-61.9％、显影剂10-39.9％。
优选的，所述固体组分，按照质量百分含量计，包括弱碱性硼硅酸盐生物活性玻璃20.1-60％、聚甲基丙烯酸甲酯(PMMA)8.5-59.7％、显影剂20-30％、过氧化苯甲酰0.2-1.5％。
优选的，所述液体组分中，甲基丙烯酸甲酯(MMA)的质量百分含量为90-99.5％，优选95-99.5％。
优选的，所述液体组分中，聚合活化剂的质量百分含量为0.5-10％，优选0.5-5％。
优选的，所述液体组分中，所述对苯二酚的含量不超过50ppm，优选30-50ppm。
优选的，所述液体组分中，按照质量百分含量计，包括甲基丙烯酸甲酯95-99.5％、N,N-二甲基对甲苯胺0.5-5％。
优选的，所述弱碱性的复合骨水泥材料中，固体组分与液体组分的质量体积比为(2.1-3.0)g：1mL；进一步优选的，所述弱碱性的复合骨水泥材料中，固体组分与液体组分的质量体积比为(2.1-2.7)g：1mL；更优选的，所述弱碱性的复合骨水泥材料中，固体组分与液体组分的质量体积比为(2.3-2.5)g：1mL。
优选的，所述固体组分为粉末状态。
优选的，所述固体组分中，所述弱碱性硼硅酸盐生物活性玻璃的粒径为30-55μm，优选40-50μm。
优选的，所述固体组分中，所述聚甲基丙烯酸甲酯的粒径为10-90μm，优选20-80μm。
本发明的第二方面提供一种弱碱性的复合骨水泥材料的制备方法。
具体的，一种弱碱性的复合骨水泥材料的制备方法，包括以下步骤：
固体组分的制备：称取制备所述弱碱性硼硅酸盐生物活性玻璃的原料组分，混合，熔融，冷却，粉碎，制得所述弱碱性硼硅酸盐生物活性玻璃，然后将所述弱碱性硼硅酸盐生物活性玻璃、聚甲基丙烯酸甲酯、显影剂混合，制得所述固体组分；
液体组分的制备：将所述甲基丙烯酸甲酯(MMA)和聚合活化剂混合，制得所述液体组分；
将所述固体组分和液体组分混合。
优选的，所述制备所述弱碱性硼硅酸盐生物活性玻璃的原料组分包括钠盐、钾盐、镁盐、钙盐、锶盐、二氧化硅、硼酸、磷酸盐。
进一步优选的，所述钠盐、钾盐、镁盐、钙盐、锶盐指钠、钾、镁、钙、锶对应的碳酸盐或碳酸氢盐。
优选的，所述固体组分制备过程中，冷却后，还包括粉碎、球磨的操作过程。
所述复合骨水泥材料在使用时，将所述固体组分和所述液体组分按照比例混合即可。
本发明的第三方面提供一种弱碱性的复合骨水泥材料的应用。
一种组织修复材料，包括上述复合骨水泥材料。
优选的，所述组织修复材料包括皮肤修复材料或骨修复材料。
以往的硼硅酸盐生物活性玻璃虽然具有好的生物活性，但是并不能往PMMA骨水泥基体中掺入很多，其中一个重要原因是硼硅酸盐生物活性玻璃的降解会造成较强的碱性，易引起生物安全性问题，诸如溶血和细胞毒性方面的问题；而较低含量的硼硅酸盐生物活性玻璃的掺入对于PMMA骨水泥的生物学效应的提高效果并不显著；因此本发明通过调整硼硅酸盐生物活性玻璃的组分，制备出弱碱性的硼硅酸盐生物活性玻璃，显著降低了其在特定溶液中浸泡并降解所引起的高碱性pH效应。
相对于现有技术，本发明的有益效果如下：
(1)本发明所述复合骨水泥材料中包括弱碱性硼硅酸盐生物活性玻璃，本发明通过调整弱碱性硼硅酸盐生物活性玻璃的组成，降低了其降解产物的pH值，达到降解后弱碱性的效 果，而弱碱性能够极高的提高细胞的存活率，并显著降低高碱性导致的溶血反应，从而显著地提高了其体外细胞相容性。
(2)虽然成骨需要一定的碱性环境，但是只有在相对合适的弱碱性条件下才能有效介导干细胞定向成骨分化、增强成骨细胞活性、并抑制破骨细胞活性；而当碱性过高时，反而会抑制干细胞的增殖分化、抑制破骨细胞的活性，并损伤宿主组织；因此鉴于碱性调控成骨理论，其碱性环境不是越高越好，而需要特定的弱碱性微环境。本发明提供的弱碱性复合骨水泥材料，恰好能提供发挥最佳成骨促进作用所需的弱碱性微环境。
(3)本发明所述复合骨水泥材料中的弱碱性硼硅酸盐生物活性玻璃含量在固体组分中的占比超过20％，例如30-60％，含量明显高于CN104922731A中骨水泥材料中的硼硅酸盐生物活性玻璃含量。故本发明可显著提高PMMA基复合骨水泥的生物活性、骨整合性和成骨再生能力等体内外生物学性能。
(4)本发明提供的复合骨水泥材料的制备方法原料易得，工艺简单，操作方便，便于应用。
附图说明
图1为实施例1-3复合骨水泥材料的力学性能；
图2为实施例1-3复合骨水泥材料、对比例1-3复合骨水泥材料对应的溶血率；
图3为实施例4-6复合骨水泥材料对应的溶血率；
图4为hBMSCs细胞在不同pH值的含有10％(质量浓度)CCK-8试剂的培养基中第1、3和7天的存活情况；
图5为hBMSCs细胞在不同pH值条件下1、3、5和7天的活细胞染色图；
图6为hBMSCs细胞在不同pH值条件下成骨诱导3、5、7与10天后的ALP染色以及ALPL基因的mRNA表达水平；
图7为hMSCs细胞在不同pH值条件下成骨诱导10天后的(a)COL1A1的免疫细胞化学；(b)相对荧光强度；(c)COL1A1基因的mRNA表达水平；(d)天狼星红染色情况；
图8为hBMSCs细胞在不同pH值条件下成骨诱导7、10和14天后的(a)茜素红染色；(b)茜素红半定量；
图9为hBMSCs细胞在不同pH值条件下成骨诱导3、7、10与14天后的成骨相关基因 SP7的mRNA表达水平。
具体实施方式
为了让本领域技术人员更加清楚明白本发明所述技术方案，现列举以下实施例进行说明。需要指出的是，以下实施例对本发明要求的保护范围不构成限制作用。
以下实施例中所用的原料、试剂或装置如无特殊说明，均可从常规商业途径得到，或者可以通过现有已知方法得到。
实施例1
一种复合骨水泥材料，包括固体组分和液体组分；
固体组分，按照质量百分含量计，包括弱碱性硼硅酸盐生物活性玻璃30％、聚甲基丙烯酸甲酯(PMMA)粉末60％、显影剂(ZrO2)10％。
液体组分，按照质量百分含量计，包括甲基丙烯酸甲酯(MMA)99.2％和N,N-二甲基对甲苯胺(DMPT)0.8％；
弱碱性硼硅酸盐生物活性玻璃的化学组成为3Na2O·2K2O·8MgO·25CaO·27B2O3·2P2O5·27SiO2·6SrO。
一种弱碱性的复合骨水泥材料的制备方法，包括以下步骤：
(1)固体组分的制备：
弱碱性硼硅酸盐生物活性玻璃的制备：
称取5.23g Na2CO3、13.64gK2CO3、38.34g(MgCO3)4·Mg(OH)2·5H2O、123.48g CaCO3、169.7g H3BO3、80.05g SiO2:、43.71g SrCO3、30.79g NaH2PO4·2H2O到容器中，搅拌混合后，倒入铂金坩埚中并放入高温炉，在1200℃下加热1小时，然后取出来直接倒在冰水里，取出玻璃物，用酒精洗一遍，放入干燥箱里烘干，烘干后放置在球磨机上球磨1小时，最后用振动筛筛选出粒径为40-45μm的弱碱性硼硅酸盐生物活性玻璃；
弱碱性硼硅酸盐生物活性玻璃占固体组分末质量百分含量为30％，将弱碱性硼硅酸盐生物活性玻璃、PMMA粉末和ZrO2置于三维混合仪中使之均匀混合(50Hz，2小时)，得到固体组分；
(2)液体组分的制备：
将甲基丙烯酸甲酯(MMA)与N,N-二甲基对甲苯胺(DMPT)混合，得到液体组分；
(3)复合骨水泥材料的制备：
在室温下，将上述固体组分与液体组分按2.4g：1mL的质量体积比混合后搅拌均匀，即可制得复合骨水泥材。
实施例2
一种弱碱性的复合骨水泥材料的制备方法，弱碱性硼硅酸盐生物活性玻璃占固体组分质量百分含量的40％，聚甲基丙烯酸甲酯(PMMA)粉末40％、显影剂(ZrO2)20％，其余过程均与实施例1相同。
实施例3
一种弱碱性的复合骨水泥材料的制备方法，弱碱性硼硅酸盐生物活性玻璃占固体组分质量百分含量的50％，聚甲基丙烯酸甲酯(PMMA)粉末40％、显影剂(ZrO2)10％，其余过程均与实施例1相同。
实施例4
制备一种弱碱性硼硅酸盐生物活性玻璃，记为：弱碱性硼硅酸盐生物活性玻璃-1，其化学组成为3Na2O·4K2O·8MgO·23CaO·27B2O3·2P2O5·27SiO2·6SrO。其制备过程为：称取5.23g Na2CO3、27.08gK2CO3、38.34g(MgCO3)4·Mg(OH)2·5H2O、112.75g CaCO3、169.7g H3BO3、80.05g SiO2:、43.71g SrCO3、30.79g NaH2PO4·2H2O到容器中，其余步骤与实施例1所描述的制备弱碱性硼硅酸盐生物活性玻璃的方法完全一致。
将本实施例制备的弱碱性硼硅酸盐生物活性玻璃替代实施例1中的弱碱性硼硅酸盐生物活性玻璃来制备复合骨水泥材料，其余步骤与实施例1完全一致。
实施例5
制备一种弱碱性硼硅酸盐生物活性玻璃，记为：弱碱性硼硅酸盐生物活性玻璃-2，其化学组成为4Na2O·6K2O·8MgO·20CaO·27B2O3·2P2O5·27SiO2·6SrO。其制备过程为：称取10.3g Na2CO3、40.29gK2CO3、38.34g(MgCO3)4·Mg(OH)2·5H2O、97.26g CaCO3、169.7g H3BO3、80.05g SiO2:、43.71g SrCO3、30.79g NaH2PO4·2H2O到容器中，其余步骤与实施例1所描述的制备弱碱性硼硅酸盐生物活性玻璃的方法完全一致。
将本实施例制备的弱碱性硼硅酸盐生物活性玻璃替代实施例1中的弱碱性硼硅酸盐生物活性玻璃来制备复合骨水泥材料，其余步骤与实施例1完全一致。
实施例6
制备一种弱碱性硼硅酸盐生物活性玻璃，记为：弱碱性硼硅酸盐生物活性玻璃-3，其化学组成为3Na2O·4K2O·8MgO·16CaO·27B2O3·2P2O5·27SiO2·13SrO。其制备过程为：称取5.23g Na2CO3、27.08gK2CO3、38.34g(MgCO3)4·Mg(OH)2·5H2O、75.96g CaCO3、169.7g H3BO3、80.05g SiO2:、91.03g SrCO3、30.79g NaH2PO4·2H2O到容器中，其余步骤与实施例1所描述的制备弱碱性硼硅酸盐生物活性玻璃的方法完全一致。
将本实施例制备的弱碱性硼硅酸盐生物活性玻璃替代实施例1中的弱碱性硼硅酸盐生物活性玻璃来制备复合骨水泥材料，其余步骤与实施例1完全一致。
对比例1
与实施例1相比，对比例1的区别仅在于使用的硼硅酸盐生物活性玻璃的组成为：6Na2O·8K2O·8MgO·16CaO·27B2O3·2P2O5·27SiO2·6SrO(称为常规硼硅酸盐生物活性玻璃)，其余过程与实施例1相同。
对比例2
与实施例2相比，对比例2的区别仅在于使用的硼硅酸盐生物活性玻璃的组成为：6Na2O·8K2O·8MgO·16CaO·27B2O3·2P2O5·27SiO2·6SrO(称为常规硼硅酸盐生物活性玻璃)，其余过程与实施例2相同。
对比例3
与实施例3相比，对比例3的区别仅在于使用的硼硅酸盐生物活性玻璃的组成为：6Na2O·8K2O·8MgO·16CaO·27B2O3·2P2O5·27SiO2·6SrO(称为常规硼硅酸盐生物活性玻璃)，其余过程与实施例3相同。
产品效果测试
1.力学性能测试
将实施例1-3的复合骨水泥材料注入模具中，在室温下固化1小时候取出，即制得长为(75±0.1)mm、宽为(10±0.1)mm、厚为(3.3±0.1)mm的复合骨水泥试样，进行四点弯曲强度、抗弯模量测试。
将实施例1-3的复合骨水泥材料注入模具中，在室温下固化1小时候取出，即制得圆柱状试样，且打磨后的尺寸达到长度为(12±0.1)mm、直径为(6±0.1)mm的复合骨水泥材 料试样，进行抗压强度测试。
图1为实施例1-3复合骨水泥材料的力学性能；图1(图1中的横坐标“弱碱性硼硅酸盐生物活性玻璃含量”表示弱碱性硼硅酸盐生物活性玻璃占复合骨水泥材料中固体组分质量百分含量依次为30％、40％、50％，即依次对应实施例1、实施例2、实施例3；图1中的虚线为YY 0459-2003中规定的标准值)中的(a)为四点弯曲强度测试结果，图1中的(b)为抗弯模量测试结果，图1中的(c)为抗压强度测试结果。
从图1可以看出，实施例1-3复合骨水泥材料试样测量的四点弯曲强度分别为54.81±1.73MPa(实施例1)、54.14±1.93MPa(实施例2)和53.29±2.01MPa(实施例3)；抗弯模量分别为强度为3766.02±83.93MPa(实施例1)、3861.51±74.19MPa(实施例2)与3866.86±62.79MPa(实施例3)；抗压强度为86.28±3.18MPa(实施例1)、90.64±2.96MPa(实施例2)与88.71±3.63MPa(实施例3)。
以上结果表明，本发明实施例1-3制备的复合骨水泥材料的力学性能良好。
对实施例4-6的复合骨水泥材料也进行同样的力学性能测试，测试结果发现力学结果皆符合YY 0459-2003中规定的标准要求，且实施例4-6之间的力学性能无显著性差异。
2.浸泡pH值测试
由实施例1-3制备的复合骨水泥材料试样，将其浸泡在生理盐水中，于37℃恒温箱中孵育2小时后，测定浸泡液的pH值，对比例1、对比例2、对比例3进行相同的实验。
实施例1-3复合骨水泥材料的浸泡液pH值分别为8.89(实施例1)、8.93(实施例2)与9.12(实施例3)，而常规硼硅酸盐生物活性玻璃复合骨水泥材料pH值为10.46(对比例1)、10.73(对比例2)与10.81(对比例3)，表明实施例1-3复合骨水泥材料试样降解后的碱性pH明显降低，优化了其在体内的生物安全性。
对实施例4-6的复合骨水泥材料也进行同样的测试，测试结果表明其与常规硼硅酸盐生物活性玻璃复合骨水泥材料的pH值相比，也具有比较低的pH值，和实施例1-3所测试的结果相似。
3.溶血测试
对实施例1-3制备的复合骨水泥材料试样进行溶血实验。对比例1-3进行相同的实验。
图2为实施例1-3复合骨水泥材料、对比例1-3复合骨水泥材料对应的溶血率。
从图2(图2中的虚线为YY/T 1651.1-2019中规定的标准值，30％、40％、50％分别指弱碱性硼硅酸盐生物活性玻璃或常规硼硅酸盐生物活性玻璃在固体组分中的含量)可以看出，实施例1-3的复合骨水泥材料的溶血率分别为1.39±0.21％(实施例1)、2.34±0.35％(实施例2)与4.69±0.29％(实施例3)，对比例1-3对应的溶血率分别为23.47±1.24％(对比例1)、42.06±2.25％(对比例2)与49.18±1.99％(对比例3)，表明实施例1-3制备的复合骨水泥材料具有远低于对比例1-3复合骨水泥材料的溶血率，具有更优异的生物安全性。
对实施例4-6制备的复合骨水泥材料试样进行同样的溶血实验。实验结果表明(图3)，实施例4-6的复合骨水泥材料的溶血率分别为1.58±0.37％(实施例4)、4.60±0.47％(实施例5)与2.67±0.33％(实施例6)，表明实施例4-6制备的复合骨水泥材料具有与实施例1-3复合骨水泥材料相当的溶血率，都具有优异的生物安全性。
4.pH对人的骨髓间充质干细胞(hBMSCs)的增殖的影响
将hBMSCs细胞以5000个/孔种于96孔板中，在不同pH值的α-MEM培养基中分别培养1、3和7天后将培养基吸出，更换含有10％(质量浓度)CCK-8试剂的培养基(不含血清)在细胞培养箱中孵育1小时，使用酶标仪在450nm检测吸光度，结果如图4所示。
图4为hBMSCs细胞在不同pH值的含有10％(质量浓度)CCK-8试剂的培养基中第1、3和7天的存活情况(图4中的“Control”表示对照组，“Absorbance@450nm”表示450nm对应的吸光度，“Time(day)”表示“时间(天)”；“*”表示p＜0.05，“**”表示p＜0.01，“***”表示p＜0.001，“****”表示p＜0.0001与Control组比较)。
从图4可知，在第1天时，所有的组的细胞活性无显著性差异。在第3天时，与对照组pH为7.350相比，pH为7.650与7.800组的吸光度无显著性差异，而pH为7.050、6.750以及7.950组的吸光度较低。在第7天时，对照组以及pH为7.650与7.800组的吸光度较第3天的明显增加，说明细胞增殖正常，未受影响。表明在pH为7.650-7.800范围内的弱碱性环境下hBMSCs细胞的增殖未受到明显影响，而pH为7.050与7.950组的吸光度与第3天的对比，增加幅度小于pH为7.350、7.650与7.800三组，并且第7天的吸光度较对照组显著降低。说明pH值大于7.800与小于7.350的条件对细胞代谢产生明显影响，细胞的增殖受到了抑制，细胞的存活率降低。而pH为6.750组的吸光度甚至小于第3天，说明在此条件下，细胞可能开始凋亡。综上，hBMSCs细胞可在pH为7.350-7.800范围内保持正常的生理代谢活动。因 此表明弱碱性环境对于细胞的生理状况至关重要。
5.碱性微环境对hBMSCs细胞存活状态的影响
将hBMSCs细胞以4*10000个/孔种于24孔板中，在不同pH值的α-MEM培养基中分别培养1、3、5和7天后将培养基吸出，加入Calcein-AM/PI试剂(活细胞死细胞双染试剂)在细胞培养箱中孵育30分钟，使用荧光显微镜观察细胞的活死状态，结果如图5所示。
图5为hBMSCs细胞在不同pH值条件下1、3、5和7天的活细胞染色图(“Control”表示对照组，“Day1”、“Day3”、“Day5”、“Day7”依次表示第1天、第3天、第5天、第7天)。
从图5可以看出，随着时间的增加，pH为7.050-7.950五组的细胞数量逐渐增多，且细胞间的排布更为致密。而pH为6.750组的细胞存活率明显下降，细胞数量明显减少，在7天时剩余活细胞数量较少。并且细胞的形态受到影响较大，细胞间的空隙增大，排布稀疏，单个细胞的形态皱缩。在1天时，pH为7.050、6.750以及7.950组对细胞的影响较大，细胞形态皱缩。pH为7.650与7.800组与对照组无明显差异。结果表明，在较生理环境偏酸性(pH＜7.050)的条件下，hBMSCs细胞的增殖受到较大程度地抑制，而生理环境微偏碱性(pH为7.650-7.800)的环境对hBMSCs细胞增殖与形态的影响较小，与生理条件相比几乎无发生明显变化。因此表明弱碱性环境有益于细胞的生长。
6.碱性微环境对hBMSCs碱性磷酸酶(ALP)表达的影响
将hBMSCs细胞成骨诱导分化3、5、7和10天后，使用碧云天BCIP/NBT碱性磷酸酶显色试剂盒检测细胞碱性磷酸酶(Alkaline phosphatase，ALP)的表达，结果如图6所示。
图6为hBMSCs细胞在不同pH值条件下成骨诱导3、5、7与10天后的ALP染色以及ALPL基因的mRNA表达水平(“Control”表示对照组，“Day3”、“Day5”、“Day7”、“Day10”依次表示第3天、第5天、第7天、第10天；“Fold change”表示差异倍数，“Time(day)”表示时间(天))。
图6中的(a)为hBMSCs细胞进行ALP染色的结果，蓝紫色的部分为ALP，颜色越深表明酶的含量越高。由图6中的(a)可见，随着成骨诱导的天数增加，各组的ALP染色的颜色加深，说明ALP的表达量都增加。在5天时，pH为7.650组的颜色较对照组更深，同样的在7天时也较对照组明显的颜色更深。而pH为7.850组的颜色与对照组相比无明显区别。 使用qPCR检测ALPL基因的mRNA表达水平(如图6中的(b)所示)，研究发现随着成骨诱导的天数增加，各组的ALPL的mRNA表达水平呈现上升趋势，这与ALP染色的结果一致。在5天时，pH为7.650的ALPL的mRNA表达水平较对照组也明显上调。结合ALP染色的结果，说明在pH为7.650的偏碱性胞外环境促进了hBMSCs细胞ALP的表达。
7.碱性微环境对hBMSCs细胞COL1A1表达的影响
将hBMSCs细胞成骨诱导分化10天后，测试COL1A1基因的mRNA表达；使用Abcam的天狼星红染液(Picro Sirius Red)进行检测，而且也使用细胞免疫荧光法对COL1A1进行检测，结果如图7所示。
图7为hMSCs细胞在不同pH值条件下成骨诱导10天后的(a)COL1A1的免疫细胞化学；(b)相对荧光强度；(c)COL1A1基因的mRNA表达水平；(d)天狼星红染色情况(图7中的“control”表示对照组，“DAPI”表示4',6-二脒基-2-苯基吲哚，“Merged”表示合并，“Relative fluorescence intensity for COL1A1staining”表示COL1A1染色的相对荧光强度，“Fold change”表示差异倍数，“Picro-Sirius Red(10days)”表示10天时，天狼星红染液；“**”表示p＜0.01，“***”表示p＜0.001)。
图7中的(a)、(b)为成骨诱导hBMSCs细胞10天时，细胞免疫荧光图及其相对荧光强度的半定量分析，绿色的部分为Ⅰ型胶原α1链(collagen type I alpha 1chain，COL1A1)蛋白。由图可知，在较生理环境偏酸性(pH为7.050)的条件下，COL1A1蛋白的表达量明显较对照组少，具有显著性差异。而在pH为7.650-7.800范围内的表达较多，并且pH为7.650组与对照组相比存在显著性差异。
从COL1A1基因的mRNA的表达水平(图7中的(c))结果可得，在pH为7.650-7.800范围内，COL1A1基因的mRNA表达较对照组显著上调，而pH为6.750、7.050以及7.950组的表达与对照组相比无明显差异。
进一步地使用天狼星红染液对hBMSCs细胞的胶原进行染色，红色的部分为胶原蛋白。结果如图7中的(d)所示，在较生理环境偏酸性的条件下(6.750-7.050)胶原的表达减少，并且发现细胞的形态与增殖情况都发生明显变化，细胞的增殖受到抑制，细胞形态皱缩。而在较生理环境碱性(7.650-7.800)的条件下，胶原的表达增加。但Ⅰ型胶原的表达并不随着碱性的增加而增加，pH为7.950组的胶原表达明显减少，同时发现细胞的形态也发生明显的 变化。结果表明，hBMSCs细胞只有在较生理微偏碱性(7.650-7.800)的条件下才促进胶原的表达，超出一定范围的pH值条件对细胞存活与细胞的形态都造成影响，从而导致在此条件下细胞的蛋白表达也受到影响。这与COL1A1基因的mRNA的表达水平的结果一致。结合COL1A1的细胞免疫荧光、mRNA的表达水平以及天狼星红染色的结果来看，表明一定范围内(pH为7.650-7.800)的碱性环境能促进hMSCs细胞Ⅰ型胶原的表达，而超出该范围的环境对细胞代谢将可能造成影响，从而降低了蛋白的表达。
8.碱性微环境对hBMSCs细胞矿化钙结节形成的影响
将hMSCs细胞成骨诱导分化3、7、10和14天后，使用索莱宝的茜素红染色液对细胞的矿化钙结节情况进行检测，结果如图8所示。
图8为hBMSCs细胞在不同pH值条件下成骨诱导7、10和14天后的(a)茜素红染色；(b)茜素红半定量(图8中的“Day”表示天，“Alizarin red S”表示茜素红染色，“control”表示对照组；“Alizarin red S Absorbance@562nm”表示茜素红染色562nm对应的吸光度，“Time(day)”表示时间(天)；“*”表示p＜0.05，“**”表示p＜0.01，“***”表示p＜0.001，“****”表示p＜0.0001与control组比较)。
从图8可以看出，在7天时，pH为7.650与7.800两组有矿化钙结节的产生，并在10天时表达量较对照组显著地提高。而在生理条件(pH为7.35-7.45)下，通常在成骨诱导分化14天时才形成矿化钙结节。在较生理微偏碱性(pH为7.650-7.800)的环境下，14天的矿化钙结节较对照组明显增加，而pH为7.950组，并无矿化钙结节的形成。其原因可能是在过碱的环境下，细胞的增殖受到影响，且不适于细胞的成骨相关蛋白如胞外基质的合成，因此无法形成矿化钙结节。此外，发现在pH＜7.350的条件下也无矿化钙结节的形成。结果表明，随着碱性的增加，矿化的形成增加，但只有在较生理环境微偏碱性(pH为7.650-7.800)的条件下能促进hBMSCs细胞矿化钙结节的形成，过碱的条件(pH大于7.950)可能对细胞的代谢产生影响。
9.碱性微环境对hBMSCs细胞成骨相关基因表达的影响
对成骨相关基因SP7的表达在成骨诱导3、7、10和14天时进行检测，结果如图9所示。
图9为hBMSCs细胞在不同pH值条件下成骨诱导3、7、10与14天后的成骨相关基因SP7的mRNA表达水平(“Flod change”表示差异倍数，“Time(day)”表示时间(天)； “*”表示p＜0.05，“**”表示p＜0.01)。
由图9可知随着天数的增加，SP7基因的表达增加。并且发现pH为7.650组的SP7基因的表达水平较对照组显著性上调。
显然，上述实施例仅仅是为清楚地说明而进行的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (12)

  1. 一种复合骨水泥材料，其特征在于，包括固体组分和液体组分；
    所述固体组分包括弱碱性硼硅酸盐生物活性玻璃、聚甲基丙烯酸甲酯、显影剂；
    所述液体组分包括甲基丙烯酸甲酯和聚合活化剂；
    所述弱碱性硼硅酸盐生物活性玻璃的化学组成为aXO·bB2O3·cP2O5·dSiO2·eY2O，其中a、b、c、d和e为摩尔份数，a为4-39，b为5-15、17-35、37-53或55-65，c为1-10，d为0-60，e为2-12，X为Ca、Mg、Sr中的一种或多种，Y为Na和/或K。
  2. 根据权利要求1所述的复合骨水泥材料，其特征在于，所述显影剂包括氧化锆和/或硫酸钡。
  3. 根据权利要求1所述的复合骨水泥材料，其特征在于，所述固体组分还包括过氧化苯甲酰。
  4. 根据权利要求1所述的复合骨水泥材料，其特征在于，所述弱碱性硼硅酸盐生物活性玻璃中，e的取值范围为5-10。
  5. 根据权利要求1所述的复合骨水泥材料，其特征在于，所述弱碱性硼硅酸盐生物活性玻璃中，a的取值范围为34-39。
  6. 根据权利要求1所述的复合骨水泥材料，其特征在于，所述弱碱性硼硅酸盐生物活性玻璃的化学组成为3Na2O·2K2O·8MgO·25CaO·27B2O3·2P2O5·27SiO2·6SrO、3Na2O·4K2O·8MgO·23CaO·27B2O3·2P2O5·27SiO2·6SrO、4Na2O·6K2O·8MgO·20CaO·27B2O3·2P2O5·27SiO2·6SrO或3Na2O·4K2O·8MgO·16CaO·27B2O3·2P2O5·27SiO2·13SrO。
  7. 根据权利要求1所述的复合骨水泥材料，其特征在于，所述弱碱性硼硅酸盐生物活性玻璃中还包括ZnO和/或CuO。
  8. 根据权利要求1-7任一项所述的复合骨水泥材料，其特征在于，所述弱碱性硼硅酸盐生物活性玻璃在所述固体组分中的质量百分含量为大于20％，小于等于60％。
  9. 根据权利要求1-7任一项所述的复合骨水泥材料，其特征在于，所述固体组分，按照质量百分含量计，包括弱碱性硼硅酸盐生物活性玻璃20.1-60％、聚甲基丙烯酸甲酯30-61.9％、 显影剂10-39.9％。
  10. 根据权利要求8所述的复合骨水泥材料，其特征在于，所述弱碱性的复合骨水泥材料中，固体组分与液体组分的质量体积比为(2.1-3.0)g：1mL。
  11. 权利要求1-10任一项所述的复合骨水泥材料的制备方法，其特征在于，包括以下步骤：
    固体组分的制备：称取制备所述弱碱性硼硅酸盐生物活性玻璃的原料组分，混合，熔融，冷却，粉碎，制得所述弱碱性硼硅酸盐生物活性玻璃，然后将所述弱碱性硼硅酸盐生物活性玻璃、聚甲基丙烯酸甲酯、显影剂混合，制得所述固体组分；
    液体组分的制备：将所述甲基丙烯酸甲酯和聚合活化剂混合，制得所述液体组分；
    将所述固体组分和液体组分混合。
  12. 一种组织修复材料，其特征在于，包括权利要求1-10任一项所述的复合骨水泥材料。
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CN104922731A (zh) * 2015-06-01 2015-09-23 深圳市中科海世御生物科技有限公司 复合骨水泥前体、锶硼酸盐生物玻璃/聚甲基丙烯酸甲酯复合骨水泥的制备方法和应用
KR20160060492A (ko) * 2014-11-20 2016-05-30 단국대학교 천안캠퍼스 산학협력단 생활성 나노복합 인산칼슘 골시멘트 조성물 및 이의 제조를 위한 키트
CN112546305A (zh) * 2020-12-10 2021-03-26 深圳先进技术研究院 一种有序多孔复合材料及其制备方法和应用
CN112791233A (zh) * 2020-12-29 2021-05-14 同济大学 生物玻璃复合骨水泥及电场处理方法
WO2022120768A1 (zh) * 2020-12-10 2022-06-16 深圳先进技术研究院 一种硼硅酸盐生物活性玻璃微纳米颗粒及制备方法和应用

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KR20160060492A (ko) * 2014-11-20 2016-05-30 단국대학교 천안캠퍼스 산학협력단 생활성 나노복합 인산칼슘 골시멘트 조성물 및 이의 제조를 위한 키트
CN104922731A (zh) * 2015-06-01 2015-09-23 深圳市中科海世御生物科技有限公司 复合骨水泥前体、锶硼酸盐生物玻璃/聚甲基丙烯酸甲酯复合骨水泥的制备方法和应用
CN112546305A (zh) * 2020-12-10 2021-03-26 深圳先进技术研究院 一种有序多孔复合材料及其制备方法和应用
WO2022120768A1 (zh) * 2020-12-10 2022-06-16 深圳先进技术研究院 一种硼硅酸盐生物活性玻璃微纳米颗粒及制备方法和应用
CN112791233A (zh) * 2020-12-29 2021-05-14 同济大学 生物玻璃复合骨水泥及电场处理方法

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