CN114767929A - Communicated porous PHBHHx bone filling microsphere material and preparation method thereof - Google Patents
Communicated porous PHBHHx bone filling microsphere material and preparation method thereof Download PDFInfo
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- 239000004005 microsphere Substances 0.000 title claims abstract description 68
- 229920001020 poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Polymers 0.000 title claims abstract description 63
- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 52
- 238000011049 filling Methods 0.000 title claims abstract description 48
- 239000000463 material Substances 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 239000000243 solution Substances 0.000 claims abstract description 52
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 50
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 48
- 239000000203 mixture Substances 0.000 claims abstract description 44
- 239000000839 emulsion Substances 0.000 claims abstract description 39
- 239000008367 deionised water Substances 0.000 claims abstract description 35
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 31
- 238000003756 stirring Methods 0.000 claims abstract description 26
- 238000010438 heat treatment Methods 0.000 claims abstract description 24
- 239000005457 ice water Substances 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 18
- 239000004094 surface-active agent Substances 0.000 claims abstract description 18
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000011259 mixed solution Substances 0.000 claims abstract description 14
- 238000002145 thermally induced phase separation Methods 0.000 claims abstract description 13
- 239000011148 porous material Substances 0.000 claims abstract description 10
- 238000000967 suction filtration Methods 0.000 claims abstract description 10
- 238000004108 freeze drying Methods 0.000 claims abstract description 9
- 238000005406 washing Methods 0.000 claims abstract description 9
- 238000005191 phase separation Methods 0.000 claims abstract description 4
- 238000004945 emulsification Methods 0.000 claims description 19
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 17
- 239000003814 drug Substances 0.000 claims description 10
- WHBMMWSBFZVSSR-UHFFFAOYSA-N 3-hydroxybutyric acid Chemical compound CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 claims description 8
- HPMGFDVTYHWBAG-UHFFFAOYSA-N 3-hydroxyhexanoic acid Chemical compound CCCC(O)CC(O)=O HPMGFDVTYHWBAG-UHFFFAOYSA-N 0.000 claims description 8
- 229940079593 drug Drugs 0.000 claims description 8
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 6
- 239000000178 monomer Substances 0.000 claims description 5
- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 claims description 4
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 4
- 238000003760 magnetic stirring Methods 0.000 claims description 4
- YPFDHNVEDLHUCE-UHFFFAOYSA-N propane-1,3-diol Chemical compound OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 210000001519 tissue Anatomy 0.000 claims description 4
- 229920001577 copolymer Polymers 0.000 claims description 3
- ZWVMLYRJXORSEP-UHFFFAOYSA-N 1,2,6-Hexanetriol Chemical compound OCCCCC(O)CO ZWVMLYRJXORSEP-UHFFFAOYSA-N 0.000 claims description 2
- 229940058015 1,3-butylene glycol Drugs 0.000 claims description 2
- DUVVGYBLYHSFMV-YGEZULPYSA-N 4-methyl-n-[[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]carbamoyl]benzamide Chemical compound C1=CC(C)=CC=C1C(=O)NC(=O)N[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DUVVGYBLYHSFMV-YGEZULPYSA-N 0.000 claims description 2
- 239000004721 Polyphenylene oxide Substances 0.000 claims description 2
- 235000019437 butane-1,3-diol Nutrition 0.000 claims description 2
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 claims description 2
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 claims description 2
- 230000001804 emulsifying effect Effects 0.000 claims description 2
- 229960005150 glycerol Drugs 0.000 claims description 2
- 238000001727 in vivo Methods 0.000 claims description 2
- 229920001467 poly(styrenesulfonates) Polymers 0.000 claims description 2
- 229920000570 polyether Polymers 0.000 claims description 2
- 229960004063 propylene glycol Drugs 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 229940006186 sodium polystyrene sulfonate Drugs 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 abstract description 12
- 230000001105 regulatory effect Effects 0.000 abstract description 3
- 230000001276 controlling effect Effects 0.000 abstract description 2
- 238000009826 distribution Methods 0.000 abstract description 2
- 230000015556 catabolic process Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- GVGUFUZHNYFZLC-UHFFFAOYSA-N dodecyl benzenesulfonate;sodium Chemical compound [Na].CCCCCCCCCCCCOS(=O)(=O)C1=CC=CC=C1 GVGUFUZHNYFZLC-UHFFFAOYSA-N 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000006116 polymerization reaction Methods 0.000 description 3
- 229940080264 sodium dodecylbenzenesulfonate Drugs 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000002639 bone cement Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000001878 scanning electron micrograph Methods 0.000 description 2
- 238000001338 self-assembly Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000001694 spray drying Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 229920002988 biodegradable polymer Polymers 0.000 description 1
- 239000004621 biodegradable polymer Substances 0.000 description 1
- 229910010293 ceramic material Inorganic materials 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- -1 fatty acid ester Chemical class 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000000399 orthopedic effect Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000011403 purification operation Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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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
- A61L27/50—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
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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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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Abstract
The invention discloses a preparation method of a communicated porous PHBHHx bone filling microsphere material, which comprises the following steps: adding PHBHHx into a mixed solution of dioxane and deionized water, heating and dissolving until the mixed solution is clear to obtain a PHBHHx solution, dripping a surfactant under the conditions of heating and stirring to emulsify the solution into emulsion, and storing the emulsion at a low temperature to perform thermally induced phase separation; storing deionized water in a refrigerator until the temperature is stable, and then putting ice blocks into the refrigerator to form an ice-water mixture; putting the emulsion stored at low temperature into an ice-water mixture and storing the emulsion in a refrigerator for two days to form a solid-liquid mixture; and (4) carrying out suction filtration and washing on the solid-liquid mixture, collecting filter residues, and carrying out freeze drying. Compared with the polymer microspheres prepared by the traditional oil/oil emulsion, the method realizes the preparation of the polymer microspheres with controllable and stable porous structures by adding the surfactant into the glycerol and combining the thermal phase separation method, and provides a new way for regulating and controlling the pore diameter and pore distribution of the porous polymer-based bone filling microspheres.
Description
Technical Field
The invention relates to a controllable prepared PHBHHx bone filling microsphere material with a communicated porous structure and a preparation method thereof, belonging to the technical field of materials science.
Background
The copolymer (PHBHHx) of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid is one of polyhydroxy fatty acid ester materials, is derived from bacteria, and can be used for preparing PHBHHx with different proportions of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid monomers by the techniques of genetic engineering and the like. Phbhfx is sustainable compared to traditional non-renewable petroleum derived polymers. Due to the characteristics of biodegradability, low irritation of degradation products and adjustable degradation rate, the PHBHHx has attracted wide attention in the fields of tissue engineering and drug delivery.
The bone filling microsphere has a high specific surface area, is a traditional medicine carrier, is also a novel tissue engineering scaffold form, and is generally made of a biological ceramic material and a biodegradable polymer material. The bone filling microspheres have various advantages in clinical application, and the microspheres can enter bone defect parts through small incisions to serve as bone scaffolds, so that minimally invasive orthopedic surgery is realized; the microspheres can be added into bone cement to improve the mechanical property of the bone cement and realize the targeted delivery of the medicament; the microspheres can enter a human body through subcutaneous injection, intravenous injection or targeted injection, and carry various medicines simultaneously, so that targeted treatment and long-acting slow release of the medicines are realized.
At present, methods for preparing the bone filling microspheres mainly comprise a spray drying method, an emulsion evaporation method, a microfluid method, a self-assembly method and a chemical polymerization method. The microspheres obtained by the spray drying method have small and uniform particle size, but the surface appearance and the internal structure of the microspheres are difficult to control in production, and the drug release and the treatment effect as a stent are limited. In the emulsion evaporation method, the structure of the microspheres can be changed by adding a third phase, but the obtained microspheres have poor particle size dispersibility, and the bone scaffold treatment effect and the drug loading capacity of the microspheres are influenced. The microspheres prepared by the microfluid method have uniform particle size and controllable form, but the preparation efficiency is low. There are limited materials that can be used for the self-assembly process. The chemical polymerization method is generally combined with the emulsion method to prepare the microspheres, but the conditions of the polymerization reaction are often extreme, the drug carrying is not facilitated, and the obtained microspheres have more impurities and need complicated purification operation. The common method for forming micro-nano communicated pores in a polymer matrix by thermally induced phase separation is adopted, but the pore morphology can be changed only by changing the concentration of a polymer solution and the temperature of thermally induced phase separation, so that the application of thermally induced phase separation to practical production is prevented.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a preparation method of PHBHHx bone filling microspheres which are based on oil/oil emulsion and thermally induced phase separation and have controllable communicated porous structures.
In order to solve the problems, the invention provides a preparation method of a communicated porous PHBHHx bone filling microsphere material, which comprises the following steps:
step 1): the PHBHHx is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, and is added into a mixed solution of dioxane and deionized water to be heated and dissolved until the mixed solution is clear, so that an internal oil phase is obtained: PHBHHx solution;
step 2): heating and dissolving a surfactant in an oil phase solvent, and stirring until the mixture is clear to obtain an external oil phase;
step 3): under the condition of heating and stirring, dripping the external oil phase prepared in the step 2) into the internal oil phase prepared in the step 1), emulsifying into emulsion, and storing the emulsion at low temperature to perform thermally induced phase separation;
and step 4): storing deionized water in a refrigerator until the temperature is stable, and then putting ice blocks into the refrigerator to form an ice-water mixture;
step 5): putting the emulsion stored at low temperature into an ice-water mixture and storing the emulsion in a refrigerator for two days to form a solid-liquid mixture;
step 6): and (4) carrying out suction filtration and washing on the solid-liquid mixture prepared in the step 5), collecting filter residues, and carrying out freeze drying to obtain the communicated porous PHBHHx bone filling microsphere material. The microsphere takes PHBHHx as a raw material, is combined with a surfactant, has a controllable pore structure, and can be applied to tissue engineering and in-vivo drug targeted delivery and long-acting delivery.
Preferably, the mole percentage of the 3-hydroxycaproic acid monomer in the PHBHHx used in the step 1) is 5-18%, and the mass average molecular weight of the 3-hydroxycaproic acid monomer is 1000-500000; the mass percentage of the deionized water in the mixed solution of the dioxane and the deionized water is not more than 30 percent.
Preferably, the heating and dissolving temperature in the step 1) is 30-150 ℃; the mass concentration of the PHBHHx solution is 0.1-20%.
Preferably, the surfactant in step 2) is at least one of sodium dodecyl sulfate, polyether F68, hexadecyl trimethyl ammonium chloride and sodium polystyrene sulfonate; the oil phase solvent is at least one of ethylene glycol, 1, 2-propylene glycol, 1, 3-propylene glycol, glycerol, 1, 3-butylene glycol, 1, 4-butylene glycol and 1,2, 6-hexanetriol.
Preferably, the mass concentration of the surfactant in the solution obtained in the step 2) is 0.001-20%.
Preferably, the mass ratio of the inner oil phase to the outer oil phase in the step 3) is 1: 1-1: 10.
Preferably, the emulsification in step 3) is ultrasonic emulsification, stirring emulsification, homogeneous emulsification or magnetic stirring emulsification.
More preferably, the rotation speed of the stirring emulsification or the magnetic stirring emulsification is 200-2000 rpm, the output power of the ultrasonic emulsification is 50-600W, and the output rotation speed of the homogeneous emulsification is 200-40000 rpm.
Preferably, the temperature of the thermal phase separation in the step 3) is-196-0 ℃.
Preferably, the temperature of the refrigerator in the steps 4) and 5) is-80-20 ℃.
The invention prepares the PHBHHx bone filling microspheres through the oil/oil emulsion. And the surfactant is added into the glycerol external phase, the type and the concentration of the surfactant are changed, and the pore diameter and the pore distribution of micropores on the PHBHHx bone filling microspheres are regulated and controlled by means of thermal phase separation. The process of regulating and controlling the pore form is simple, and the prepared bone filling microsphere has good biocompatibility and biodegradability. The PHBHHx bone filling microsphere is expected to be used as a bone scaffold and a drug carrier to be applied to clinic.
Drawings
FIG. 1 is an SEM photograph of freeze-dried PHBHHx bone filling microspheres prepared in example 1.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.
Example 1
A method for preparing biodegradable controllable communicated porous PHBHHx bone filling microspheres is an example of preparing the PHBHHx porous bone filling microspheres by using a surfactant sodium dodecyl sulfate, and comprises the following steps:
the method comprises the following steps: adding 0.1g of PHBHHx into 4g of dioxane/deionized water mixed solution, heating and dissolving until the mixture is clear to obtain a polymer solution with the mass concentration percentage of 2.5%, wherein the mass ratio of dioxane to deionized water is 9: 1;
step two: adding 0.08g of sodium dodecyl sulfate into 8g of glycerol, heating in a water bath at 60 ℃, magnetically stirring at 1000rpm, and changing the solution from turbid to clear to obtain a sodium dodecyl sulfate/glycerol solution with the mass percent of 1%;
step three: heating and stirring the clear solution prepared in the first step, dripping the clear solution prepared in the second step into the heated and stirred clear solution, and continuously stirring the solution for 10min to form emulsion;
step four: standing the emulsion prepared in the third step in a refrigerator with the temperature of-20 ℃ for 4 hours, and carrying out thermally induced phase separation;
step five: storing 200mL of deionized water in a refrigerator with the temperature set to 4 ℃ for 4 h;
step six: putting ice blocks into the deionized water prepared in the fifth step to form an ice-water mixture;
step seven: putting the emulsion treated in the fifth step into the ice-water mixture prepared in the sixth step, and storing the emulsion in a refrigerator with the temperature set to be 4 ℃ for two days to form a solid-liquid mixture;
step eight: and (4) carrying out suction filtration and washing on the solid-liquid mixture prepared in the step seven, collecting filter residues, and carrying out freeze drying (-40 ℃, 30Pa) to obtain the PHBHHx bone filling microspheres.
FIG. 1 is an SEM image of the PHBHHx bone-filling microspheres obtained in example 1, and it can be seen from the SEM image that the bone-filling microspheres have a diameter of 100-200 μm, interconnected porous structures and uniform pore sizes. The porous structure increases the specific surface area of the microsphere, and is beneficial to the differentiation and proliferation of cells on the microsphere and the entry of the cells into the stent. The porous structure is also beneficial to drug release and specific carrying.
Example 2
A method for preparing biodegradable controllable communicated porous PHBHHx bone filling microspheres is an example of preparing the PHBHHx porous bone filling microspheres by using a surfactant of sodium dodecyl benzene sulfonate, and comprises the following steps:
the method comprises the following steps: adding 0.1g of PHBHHx into 4g of dioxane/deionized water mixed solution, heating and dissolving until the mixture is clear to obtain a polymer solution with the mass concentration percentage of 2.5%, wherein the mass ratio of dioxane to deionized water is 9: 1;
step two: adding 0.08g of sodium dodecyl benzene sulfonate into 8g of glycerol, heating in a water bath at 60 ℃, magnetically stirring at 1000rpm, and changing the turbid solution into a clear solution to obtain a sodium dodecyl benzene sulfonate/glycerol solution with the mass percent of 1%;
step three: heating and stirring the clear solution prepared in the first step, dripping the clear solution prepared in the second step into the heated and stirred clear solution, and continuously stirring the solution for 10min to form emulsion;
step four: standing the emulsion prepared in the step three in a refrigerator with the temperature set to-20 ℃ for 4 hours, and carrying out thermally induced phase separation;
step five: storing 200mL of deionized water in a refrigerator with the temperature set to 4 ℃ for 4 h;
step six: putting ice blocks into the deionized water prepared in the fifth step to form an ice-water mixture;
step seven: putting the emulsion treated in the fifth step into the ice-water mixture prepared in the sixth step, and storing the emulsion in a refrigerator with the temperature set to be 4 ℃ for two days to form a solid-liquid mixture;
step eight: and (4) carrying out suction filtration and washing on the solid-liquid mixture prepared in the step seven, collecting filter residues, and carrying out freeze drying (-40 ℃, 30Pa) to obtain the PHBHHx bone filling microspheres.
Example 3
A method for preparing biodegradable controllable communicated porous PHBHHx bone filling microspheres is an example of preparing the PHBHHx porous bone filling microspheres by using a surfactant sodium dodecyl sulfate, and comprises the following steps:
the method comprises the following steps: adding 0.08g of PHBHHx into 4g of dioxane/deionized water mixed solution, heating and dissolving until the solution is clear to obtain a polymer solution with the mass concentration percentage of 2.0%, wherein the mass ratio of dioxane to deionized water is 9: 1;
step two: adding 0.08g of sodium dodecyl sulfate into 8g of glycerol, heating in a water bath at 60 ℃, magnetically stirring at 1000rpm, and changing the solution from turbid to clear to obtain a sodium dodecyl sulfate/glycerol solution with the mass percent of 1%;
step three: heating and stirring the clear solution prepared in the first step, dripping the clear solution prepared in the second step into the clear solution, and continuously stirring the clear solution for 10min to form emulsion;
step four: standing the emulsion prepared in the third step in a refrigerator with the temperature of-20 ℃ for 4 hours, and carrying out thermally induced phase separation;
step five: storing 200mL of deionized water in a refrigerator with the temperature set to 4 ℃ for 4 h;
step six: putting the ice blocks into the deionized water prepared in the fifth step to form an ice-water mixture;
step seven: putting the emulsion treated in the fifth step into the ice-water mixture prepared in the sixth step, and storing the mixture in a refrigerator with the temperature set to be 4 ℃ for two days to form a solid-liquid mixture;
step eight: and (4) carrying out suction filtration and washing on the solid-liquid mixture prepared in the seventh step, collecting filter residues, and carrying out freeze drying (-40 ℃, 30Pa) to obtain the PHBHHx bone filling microspheres.
Example 4
A method for preparing biodegradable controllable communicated porous PHBHHx bone filling microspheres is an example of preparing the PHBHHx porous bone filling microspheres by using a surfactant sodium dodecyl sulfate, and comprises the following steps:
the method comprises the following steps: 0.1g of PHBHHx is added into 4g of dioxane/deionized water mixed solution, heated and dissolved until the solution is clear, and a polymer solution with the mass concentration percentage of 2.5% is obtained, wherein the mass ratio of dioxane to deionized water is 9: 1.
Step two: 0.08g of sodium dodecyl sulfate was added to 8g of glycerol and heated in a water bath at 60 ℃ and magnetically stirred at 1000rpm, the solution changed from cloudy to clear, giving a 1% by weight sodium dodecyl sulfate/glycerol solution.
Step three: heating and stirring the clear solution prepared in the step one, simultaneously dripping the clear solution prepared in the step two, and continuously stirring for 10min to form emulsion.
Step four: and standing the emulsion prepared in the step three for 4 hours in a refrigerator with the temperature set to be-60 ℃, and carrying out thermally induced phase separation.
Step five: 200mL of deionized water was stored in a refrigerator set at 4 ℃ for 4 h.
Step six: and putting the ice blocks into the deionized water prepared in the step five to form an ice-water mixture.
Step seven: and (4) putting the emulsion treated in the fifth step into the ice-water mixture prepared in the sixth step, and storing the mixture in a refrigerator with the temperature set to be 4 ℃ for two days to form a solid-liquid mixture.
Step eight: and (4) carrying out suction filtration and washing on the solid-liquid mixture prepared in the step seven, collecting filter residues, and carrying out freeze drying (-40 ℃, 30Pa) to obtain the PHBHHx bone filling microspheres.
Example 5
A method for preparing biodegradable controllable communicated porous PHBHHx bone filling microspheres is an example of preparing the PHBHHx porous bone filling microspheres by using a surfactant sodium dodecyl sulfate, and comprises the following steps:
the method comprises the following steps: adding 0.1g of PHBHHx into 4g of dioxane/deionized water mixed solution, heating and dissolving until the mixture is clear to obtain a polymer solution with the mass concentration percentage of 2.5%, wherein the mass ratio of dioxane to deionized water is 9: 1;
step two: adding 0.08g of sodium dodecyl sulfate into 8g of glycerol, heating in a water bath at 60 ℃, magnetically stirring at 1000rpm, and changing the solution from turbid to clear to obtain a sodium dodecyl sulfate/glycerol solution with the mass percent of 1%;
step three: heating and stirring the clear solution prepared in the first step, dripping the clear solution prepared in the second step into the heated and stirred clear solution, and continuously stirring the solution for 10min to form emulsion;
step four: standing the emulsion prepared in the third step in a refrigerator with the temperature of-20 ℃ for 4 hours, and carrying out thermally induced phase separation;
step five: storing 200mL of deionized water in a refrigerator with the temperature set to 4 ℃ for 4 h;
step six: putting ice blocks into the deionized water prepared in the fifth step to form an ice-water mixture;
step seven: putting the emulsion treated in the fifth step into the ice-water mixture prepared in the sixth step, and storing the emulsion in a refrigerator with the temperature set to be 4 ℃ for two days to form a solid-liquid mixture;
step eight: and (4) carrying out suction filtration and washing on the solid-liquid mixture prepared in the step seven, collecting filter residues, and carrying out freeze drying (-40 ℃, 30Pa) to obtain the PHBHHx bone filling microspheres.
Example 6
A method for preparing biodegradable controllable communicated porous PHBHHx bone filling microspheres is an example of preparing the PHBHHx porous bone filling microspheres by using a surfactant sodium dodecyl sulfate, and comprises the following steps:
the method comprises the following steps: adding 0.1g of PHBHHx into 4g of dioxane/deionized water mixed solution, heating and dissolving until the mixture is clear to obtain a polymer solution with the mass concentration percentage of 2.5%, wherein the mass ratio of dioxane to deionized water is 8: 2;
step two: adding 0.08g of sodium dodecyl sulfate into 8g of glycerol, heating in a water bath at 60 ℃, magnetically stirring at 1000rpm, and changing the solution from turbid to clear to obtain a sodium dodecyl sulfate/glycerol solution with the mass percent of 1%;
step three: heating and stirring the clear solution prepared in the first step, dripping the clear solution prepared in the second step into the clear solution, and continuously stirring the clear solution for 10min to form emulsion;
step four: standing the emulsion prepared in the third step in a refrigerator with the temperature of-20 ℃ for 4 hours, and carrying out thermally induced phase separation;
step five: storing 200mL of deionized water in a refrigerator with the temperature set to 4 ℃ for 4 h;
step six: putting ice blocks into the deionized water prepared in the fifth step to form an ice-water mixture;
step seven: putting the emulsion treated in the fifth step into the ice-water mixture prepared in the sixth step, and storing the emulsion in a refrigerator with the temperature set to be 4 ℃ for two days to form a solid-liquid mixture;
step eight: and (4) carrying out suction filtration and washing on the solid-liquid mixture prepared in the step seven, collecting filter residues, and carrying out freeze drying (-40 ℃, 30Pa) to obtain the PHBHHx bone filling microspheres.
Example 7
A method for preparing biodegradable, controllably-communicated porous phbhfx bone filler microspheres, this example being an in vitro degradation experiment using phbhfx bone filler microspheres prepared according to example 1, comprising the following steps:
the method comprises the following steps: respectively adding 0.05g of PHBHHx bone filling microspheres into a plurality of beakers filled with 100mL of PBS, and performing shake culture at 37 ℃;
step two: at fixed time points (12h, 1d, 2d, 3d, 5d, 7d, 14d and 28d) a beaker was removed, washed with suction filtration using deionized water, and the solids were collected and lyophilized;
step three: weighing the solid residual mass m2And calculating the degradation rate, wherein the formula is as follows:
wherein m is1Is the initial mass of the microspheres.
Claims (10)
1. A preparation method of a communicated porous PHBHHx bone filling microsphere material is characterized by comprising the following steps:
step 1): the PHBHHx is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, and is added into a mixed solution of dioxane and deionized water to be heated and dissolved until the mixed solution is clear, so that an internal oil phase is obtained: PHBHHx solution;
step 2): heating and dissolving a surfactant in an oil phase solvent, and stirring until the mixture is clear to obtain an external oil phase;
step 3): under the condition of heating and stirring, dripping the external oil phase prepared in the step 2) into the internal oil phase prepared in the step 1), emulsifying into emulsion, and storing the emulsion at low temperature to perform thermally induced phase separation;
step 4): storing deionized water in a refrigerator until the temperature is stable, and then putting ice blocks into the refrigerator to form an ice-water mixture;
and step 5): putting the emulsion stored at low temperature into an ice-water mixture and storing the emulsion in a refrigerator for two days to form a solid-liquid mixture;
step 6): and (4) carrying out suction filtration and washing on the solid-liquid mixture prepared in the step 5), collecting filter residues, and carrying out freeze drying to obtain the communicated porous PHBHHx bone filling microsphere material. The microsphere takes PHBHHx as a raw material, combines with a surfactant, has a controllable pore structure, and can be applied to tissue engineering and in-vivo drug targeted delivery and long-acting delivery.
2. The method for preparing the communicated porous PHBHHx bone filling microsphere material of claim 1, wherein in the PHBHHx used in the step 1), the mole percentage of 3-hydroxyhexanoic acid monomer is 5-18%, and the mass average molecular weight of the 3-hydroxyhexanoic acid monomer is 1000-500000; the mass percent of the deionized water in the mixed solution of the dioxane and the deionized water is not more than 30 percent.
3. The preparation method of the communicated porous PHBHHx bone filling microsphere material as claimed in claim 1, wherein the temperature for heating and dissolving in the step 1) is 30-150 ℃; the mass concentration of the PHBHHx solution is 0.1-20%.
4. The method for preparing the interconnected porous PHBHHx bone filling microsphere material of claim 1, wherein the surfactant in step 2) is at least one of sodium dodecyl sulfate, polyether F68, cetyltrimethylammonium chloride and sodium polystyrene sulfonate; the oil phase solvent is at least one of ethylene glycol, 1, 2-propylene glycol, 1, 3-propylene glycol, glycerol, 1, 3-butylene glycol, 1, 4-butylene glycol and 1,2, 6-hexanetriol.
5. The preparation method of the communicated porous PHBHHx bone filling microsphere material as claimed in claim 1, wherein the mass concentration of the surfactant in the solution obtained in the step 2) is 0.001-20%.
6. The preparation method of the communicated porous PHBHHx bone filling microsphere material as claimed in claim 1, wherein the mass ratio of the inner oil phase to the outer oil phase in the step 3) is 1: 1-1: 10.
7. The method for preparing the communicated porous PHBHHx bone filling microsphere material of claim 1, wherein the emulsification in the step 3) is ultrasonic emulsification, stirring emulsification, homogeneous emulsification or magnetic stirring emulsification.
8. The preparation method of the communicated porous PHBHHx bone filling microsphere material of claim 7, wherein the rotation speed of stirring emulsification or magnetic stirring emulsification is 200-2000 rpm, the output power of ultrasonic emulsification is 50-600W, and the output rotation speed of homogeneous emulsification is 200-40000 rpm.
9. The preparation method of the communicated porous PHBHHx bone filling microsphere material of claim 1, wherein the temperature of the thermal phase separation in the step 3) is-196-0 ℃.
10. The preparation method of the communicated porous PHBHHx bone filling microsphere material as claimed in claim 1, wherein the temperature of the refrigerator in the steps 4) and 5) is-80 to 20 ℃.
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