WO2020233176A1 - 生长因子缓释微球、组织工程软骨复合支架及制备方法 - Google Patents

生长因子缓释微球、组织工程软骨复合支架及制备方法 Download PDF

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WO2020233176A1
WO2020233176A1 PCT/CN2020/075159 CN2020075159W WO2020233176A1 WO 2020233176 A1 WO2020233176 A1 WO 2020233176A1 CN 2020075159 W CN2020075159 W CN 2020075159W WO 2020233176 A1 WO2020233176 A1 WO 2020233176A1
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growth factor
microspheres
aqueous solution
release
sustained
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PCT/CN2020/075159
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English (en)
French (fr)
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郭全义
杨振
卢世璧
刘舒云
眭翔
黄靖香
郭维民
田广招
孙志强
查康康
周建
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中国人民解放军总医院
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Publication of WO2020233176A1 publication Critical patent/WO2020233176A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/602—Type of release, e.g. controlled, sustained, slow
    • A61L2300/604—Biodegradation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/62—Encapsulated active agents, e.g. emulsified droplets
    • A61L2300/622—Microcapsules
    • 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 application belongs to the technical field of medical materials, and specifically relates to a growth factor sustained-release microsphere, a tissue engineering cartilage composite scaffold and a preparation method.
  • Cartilage is an important part of the skeletal system in humans or animals.
  • Cartilage tissue is a highly specialized connective tissue. It is characterized by the lack of blood vessels, nerves and lymphatic vessels in the tissue. The extracellular matrix is dense and solid.
  • Cartilage cells are composed of a large number of cells. The outer matrix (such as collagen, fiber, protein, polysaccharide, etc.) is surrounded. It is precisely due to the histological characteristics of cartilage, the composition of biochemical components and the characteristics of low metabolism and low proliferation of cartilage cells that limit the proliferative response and migration of chondrocytes to the injured area, leading to self-repair and self-repair of cartilage tissue after injury. The ability to regenerate is low, and it is difficult to repair and regenerate itself, and it will gradually increase, leading to osteoarthritis and joint dysfunction.
  • this application provides a growth factor sustained-release microsphere, which includes a poly(lactic-co-glycolic acid) (PLGA) matrix material and a recombinant encapsulated in the matrix material Human Transforming Growth Factor ⁇ 3 (Recombinant Human Transforming Growth Factor-beta 3, referred to as rhTGF- ⁇ 3); the encapsulation amount of rhTGF- ⁇ 3 in the microspheres is 5ng/mg ⁇ 200ng/mg; the sustained release period of the microspheres is more than 28 days .
  • PLGA poly(lactic-co-glycolic acid)
  • this application provides a method for preparing growth factor sustained-release microspheres, which includes the following steps:
  • rhTGF- ⁇ 3 aqueous solution with a concentration of 1 ⁇ g/mL ⁇ 100 ⁇ g/mL;
  • PVA polyvinyl alcohol
  • the rhTGF- ⁇ 3 aqueous solution and the PLGA oil phase solution were ultrasonically mixed in an ice bath to obtain a primary emulsion;
  • the microsphere solution is separated and processed, and the initial microspheres are washed and freeze-dried to obtain growth factor sustained-release microspheres.
  • the encapsulation amount of rhTGF- ⁇ 3 in the microspheres is 5ng/mg ⁇ 200ng/mg.
  • the period is more than 28 days.
  • this application provides a tissue engineered cartilage composite scaffold, which includes:
  • the tissue engineering scaffold includes a plurality of scaffold layers sequentially stacked along its height direction.
  • One of the two adjacent scaffold layers includes a plurality of fiber groups spaced apart from each other along the first direction, and the other includes a plurality of fiber groups along the second The fiber groups are spaced apart in each direction.
  • Each fiber group includes a degradable polymer fiber and a hydrogel fiber arranged on one or both sides of the degradable polymer fiber's own width direction.
  • the hydrogel fiber runs along the degradable polymer fiber. The length direction of the fiber extends, and the first direction intersects the second direction;
  • the matrix material is compounded in the hydrogel fibers of the tissue engineering scaffold
  • the aforementioned growth factor sustained-release microspheres are compounded in the hydrogel fibers of the tissue engineering scaffold.
  • the present application provides a method for preparing a tissue engineered cartilage composite scaffold, which includes the following steps:
  • the above-mentioned tissue engineering cartilage composite scaffold is printed.
  • the growth factor sustained-release microspheres, tissue engineered cartilage composite scaffold and preparation method provided in the present application wherein the growth factor sustained-release microspheres have the characteristics of good rhTGF- ⁇ 3 controlled and sustained release performance, and can achieve long-term effective rhTGF- ⁇ 3 release concentration. And long-term effective use of rhTGF- ⁇ 3 cell recruitment ability to improve the regeneration and repair of defective cartilage tissue.
  • the tissue-engineered cartilage composite scaffold is a good biomimetic of the microenvironment of cartilage collagen orientation and component distribution and cell growth, fusion of the good mechanical properties and biodegradability of the degradable polymer fiber, and good biocompatibility of the matrix material
  • the controllable slow-release performance and strong cell recruitment ability of the growth factor slow-release microspheres are beneficial to the growth, migration, proliferation and redifferentiation of the cells, which can well promote the partial or all defective cartilage in the avascular zone.
  • the regeneration and repair of tissues make the new cartilage tissue have excellent morphology, mechanical properties and physiological functions.
  • Figure 1 is a scanning electron microscope (SEM) image of the growth factor sustained-release microspheres of Example 1 of the application.
  • Example 2 is a scanning electron microscope (SEM) image of the growth factor sustained-release microspheres of Example 2 of the application.
  • Figure 3 is a scanning electron microscope (SEM) image of the growth factor sustained-release microspheres of Example 3 of the application.
  • Figure 5 is an image of the results of the Transwell migration experiment, where A is the negative group; B is the positive group; C is the blank group; D is the experimental group 1; E is the experimental group 2; F is the experimental group 3.
  • Figure 6 is a statistical analysis diagram of the results of the Transwell migration experiment, where A is the negative group; B is the positive group; C is the blank group; D is the experimental group 1; E is the experimental group 2; F is the experimental group 3.
  • Fig. 7 is a structural diagram of a tissue engineering cartilage composite scaffold according to an embodiment of the application.
  • Figure 8 is a schematic cross-sectional view of a tissue engineered cartilage composite scaffold according to an embodiment of the application.
  • FIG. 9 is a schematic diagram of the scaffold layer in the tissue engineering cartilage composite scaffold according to an embodiment of the application.
  • Fig. 10 is an ordinary optical microscope image of the tissue engineering cartilage composite scaffold according to an embodiment of the application.
  • Figure 11 is a confocal microscope image of a tissue engineered cartilage composite scaffold loaded with rabbit adipose stem cells according to an embodiment of the application.
  • Figure 12 is an SEM image of mouse macrophage RAW cells loaded on a tissue engineered cartilage composite scaffold according to an example of the application.
  • any lower limit may be combined with any upper limit to form an unspecified range; and any lower limit may be combined with other lower limits to form an unspecified range, and any upper limit may be combined with any other upper limit to form an unspecified range.
  • every point or single value between the end points of the range is included in the range. Therefore, each point or single numerical value can be used as its own lower limit or upper limit in combination with any other point or single numerical value or in combination with other lower or upper limits to form an unspecified range.
  • Tissue engineering is a comprehensive application of the principles and techniques of engineering and life sciences.
  • a biologically active implant is constructed in vitro and then implanted in the body to achieve the purpose of repairing tissue defects and rebuilding tissue functions.
  • the embodiments of the present application provide a growth factor sustained-release microsphere, a tissue engineered cartilage composite scaffold and a preparation method.
  • the microsphere includes a PLGA matrix material and rhTGF- ⁇ 3 encapsulated in the matrix material; the encapsulation amount of rhTGF- ⁇ 3 in the microsphere is 5ng/mg ⁇ 200ng/mg; the sustained release period of microspheres is more than 28 days.
  • the growth factor sustained-release microspheres provided by the examples of the application have the characteristics of good rhTGF- ⁇ 3 controlled and slow-release performance, can achieve long-term effective rhTGF- ⁇ 3 release concentration, and long-term effective use of rhTGF- ⁇ 3 cell recruiting ability to achieve improved Regeneration and repair effect of defective cartilage tissue.
  • PLGA is used to encapsulate rhTGF- ⁇ 3 to achieve long-term sustained release of rhTGF- ⁇ 3, avoiding the short-acting time, easy degradation and transient effects of rhTGF- ⁇ 3, and effectively preventing rhTGF- ⁇ 3 from metabolic processes and physiological environments Change, or denature with enzymes, to better play the biological role of rhTGF- ⁇ 3.
  • the growth factor sustained-release microspheres provided in the examples of the application can be used in a tissue engineered cartilage composite scaffold to achieve long-term effective regeneration and repair effects on defective cartilage tissue.
  • the encapsulation amount of rhTGF- ⁇ 3 in the growth factor sustained-release microspheres is 30ng/mg-200ng/mg.
  • the sustained release amount of the microspheres on the first day is 0.05% to 0.5%, and the microspheres have the maximum sustained release rate on the first day.
  • the sustained release amount of the microspheres in the first week is 0.1% to 1%.
  • sustained release amount for the first day and “sustained release amount for the first week” are calculated based on the initial rhTGF- ⁇ 3 content of the microspheres.
  • the sustained release period of the growth factor sustained-release microspheres can reach more than half a year, such as 200 days to 600 days, and for example, 300 days to 600 days.
  • the molecular weight of PLGA is preferably 1 ⁇ 10 5 Da to 1.1 ⁇ 10 5 Da, more preferably 1.02 ⁇ 10 5 Da.
  • the molar ratio of lactic acid to glycolic acid in PLGA is preferably 1:1 to 5:1, such as 2.5:1 to 3.5:1, and further, for example, 3:1.
  • the particle size of the growth factor sustained-release microspheres is preferably 5 ⁇ m to 100 ⁇ m, more preferably 5 ⁇ m to 50 ⁇ m. This is conducive to better control and slow release of rhTGF- ⁇ 3.
  • the surface of the growth factor sustained-release microspheres may have slight undulations, and the distance between two adjacent wave crests is, for example, 0.1 ⁇ m to 2.5 ⁇ m, and further 0.3 ⁇ m to 2 ⁇ m.
  • the growth factor sustained-release microspheres have a good microsphere appearance, which is conducive to cell adhesion, promotes cell growth, migration, proliferation and redifferentiation, and improves the regeneration and repair effects of defective cartilage tissue.
  • the second aspect of the embodiments of the present application provides a method for preparing growth factor sustained-release microspheres, by which the above-mentioned growth factor sustained-release microspheres can be prepared.
  • the preparation method includes the following steps:
  • rhTGF- ⁇ 3 may be dissolved in deionized water to prepare an aqueous solution of rhTGF- ⁇ 3 with a concentration of 1 ⁇ g/mL-100 ⁇ g/mL as the internal water phase w 1 .
  • step S20 PLGA can be dissolved in dichloromethane (DCM) to prepare a PLGA oil phase solution with a concentration of 20 mg/mL to 25 mg/mL as the oil phase o.
  • This kind of oil phase o can have a higher encapsulation efficiency for rhTGF- ⁇ 3.
  • the molecular weight of PLGA is preferably 1 ⁇ 10 5 Da to 1.1 ⁇ 10 5 Da, more preferably 1.02 ⁇ 10 5 Da. Further, the molar ratio of lactic acid to glycolic acid in PLGA is preferably 1:1 to 5:1, such as 2.5:1 to 3.5:1, and further, for example, 3:1.
  • the effect between rhTGF- ⁇ 3 and these PLGAs is good, which can increase the encapsulation rate of rhTGF- ⁇ 3 by the growth factor sustained-release microspheres, and make the growth factor sustained-release microspheres have an improved rhTGF- ⁇ 3 controlled slow release rate. Extend the sustained-release period to meet the long-term regeneration and repair needs of cartilage defects.
  • these PLGAs have good biodegradability and can provide excellent carriers for cell attachment, proliferation and differentiation.
  • S30 Provide an aqueous PVA solution with a concentration of 5 mg/mL to 10 mg/mL.
  • step S30 PVA is dissolved in deionized water to prepare an aqueous PVA solution with a concentration of 5 mg/mL-10 mg/mL as the outer water phase w 2 .
  • the external water phase w 2 can provide an excellent environment for the formation of growth factor sustained-release microspheres.
  • the molecular weight of the polyvinyl alcohol in the PVA aqueous solution is preferably 6 ⁇ 10 4 Da to 7 ⁇ 10 4 Da, more preferably 6.7 ⁇ 10 4 Da.
  • step S40 by ultrasonically mixing the rhTGF- ⁇ 3 aqueous solution and the PLGA oil phase solution in an ice bath, they can be fully homogenized and emulsified, which is beneficial to increase the encapsulation efficiency of PLGA on rhTGF- ⁇ 3, and to improve the rhTGF- ⁇ 3 in micro The uniformity of dispersion in the ball, thereby increasing the utilization rate of rhTGF- ⁇ 3, and improving the controlled and slow release effect of rhTGF- ⁇ 3. In addition, the biological activity of rhTGF- ⁇ 3 is effectively preserved.
  • step S40 the mixing volume ratio of the rhTGF- ⁇ 3 aqueous solution and the PLGA oil phase solution is preferably 1:5 to 1:10.
  • the ultrasonic power of the ultrasonic mixing is preferably 70W-75W, and the ultrasonic time is preferably 10s-30s.
  • Step S40 can be performed in an ultrasonic cell crusher, such as the US Qsonica Q125 ultrasonic crusher.
  • the probe of the crusher is oscillated at 60% amplitude (ultrasonic power is 75W) for 10s to 30s, and the rhTGF- ⁇ 3 aqueous solution and the PLGA oil phase solution are sonicated. Mix to obtain a primary emulsion.
  • step S50 rhTGF- ⁇ 3 and PLGA self-assemble into microspheres, and the DCM is also volatilized and removed during the stirring process.
  • the mixing volume ratio of the primary emulsion and the PVA aqueous solution may be 1:5 to 1:25, for example, 1:8 to 1:12.
  • the speed of stirring and mixing is preferably 500 rpm to 1000 rpm, and the time is preferably 4 h to 12 h.
  • the microsphere solution can be separated and processed to obtain initial microspheres by a method such as suction filtration, centrifugal separation, and the like.
  • the microsphere solution is centrifuged at 1500 rpm to 2000 rpm for 3 min to 5 min.
  • the initial microspheres can be washed with deionized water for 2 to 4 times, such as 3 times.
  • the washed initial microspheres can be freeze-dried at -4 to -20°C for 18 hours to 24 hours to obtain growth factor sustained-release microspheres.
  • the prepared growth factor sustained-release microspheres are stored at a temperature of -4°C to -25°C for use, for example, at -20°C for use.
  • the preparation method of the growth factor sustained-release microspheres of the embodiments of the present application also has a relatively high encapsulation efficiency for rhTGF- ⁇ 3, which can reach more than 80%, and furthermore, more than 90%, which can improve the utilization rate of rhTGF- ⁇ 3.
  • the tissue engineering cartilage composite scaffold includes a tissue engineering scaffold and a matrix material compounded with the tissue engineering scaffold and the aforementioned growth Factor sustained-release microspheres.
  • the tissue engineering scaffold is a porous frame structure, which includes a plurality of scaffold layers 1 stacked in sequence along its height direction.
  • One of the two adjacent scaffold layers 1 includes a plurality of scaffold layers along the first direction (as shown in the X direction in FIG. )
  • Fiber groups 2 arranged at intervals
  • the other includes a plurality of fiber groups 2 arranged at intervals along a second direction (as shown in FIG. 8 perpendicular to the paper)
  • each fiber group 2 includes degradable polymer fibers 3 and the hydrogel fibers 4 arranged on one or both sides of the degradable polymer fiber's own width direction.
  • the hydrogel fibers extend along the length direction of the degradable polymer fiber, wherein the first direction intersects the second direction, Preferably, the first direction is perpendicular to the second direction.
  • Both the matrix material and the growth factor slow-release microspheres are compounded in the hydrogel fibers 4 of the tissue engineering scaffold.
  • the tissue engineering cartilage composite scaffold provided by the examples of this application is a good biomimetic of the cartilage collagen orientation and component distribution and the microenvironment of cell growth, and incorporates the degradable polymer fiber. 3 Good mechanical properties and biodegradability, and good matrix materials The biocompatibility, the controlled slow-release performance of the growth factor sustained-release microspheres and the strong cell recruitment ability make the composite scaffold have good biocompatibility, mechanical properties, degradability, and low immunogenicity It has sustained release of growth factors and can recruit endogenous stem cells to regenerate and repair defective cartilage tissues, promote cell growth, migration, proliferation and redifferentiation, thereby promoting the regeneration of partial or all defective cartilage tissues in the avascular zone. Repair, so that the new cartilage tissue has excellent morphology, mechanical properties and physiological functions.
  • the interval between adjacent fiber groups 2 is preferably 300 ⁇ m to 500 ⁇ m; the interval between degradable polymer fibers 3 in adjacent fiber groups 2 is preferably 750 ⁇ m to 1500 ⁇ m;
  • the height of the scaffold layer 1 is preferably 100 ⁇ m to 500 ⁇ m.
  • This kind of tissue engineered cartilage composite scaffold better bionics the oriented macroporous structure of cartilage tissue and the small pore structure inside the macroporous structure, so that the tissue engineered cartilage composite scaffold has higher mechanical properties and biocompatibility. At the same time, it can also make the endogenous stem cells easier to migrate, and complete the proliferation and differentiation during the migration process, and realize the regeneration and repair of some cartilage defects or all cartilage defects, which has a higher repair effect.
  • the width of the degradable polymer fiber is preferably 200 ⁇ m to 800 ⁇ m; the width of the hydrogel fiber is preferably 200 ⁇ m to 800 ⁇ m.
  • the material of the degradable polymer fiber can be selected from polycaprolactone PCL, polyurethane PU, polylactic acid PLA, polylactic acid-glycolic acid copolymer PLGA, polylactic acid-polycaprolactone copolymer PCLA, polyamino acid PAA and polyglycolic acid One or more of PGA.
  • PCL which has good mechanical properties and biodegradability.
  • the molecular weight of PCL is preferably 2 ⁇ 10 4 Da to 5 ⁇ 10 5 Da, such as 2 ⁇ 10 4 Da to 1 ⁇ 10 5 Da.
  • the hydrogel fibers are preferably methacrylated hydrogel (GelMA) fibers with a degree of methylation of 40% to 80%, more preferably GelMA with a degree of methylation of 50% to 70% fiber.
  • This kind of hydrogel fiber has good printability.
  • the use of this kind of hydrogel fiber is beneficial to cell attachment, migration, proliferation and differentiation.
  • the matrix material can be selected from one or more of acellular cartilage extracellular matrix, type I collagen, type II collagen, bacterial cellulose, silk protein and glycosaminoglycans, for example, acellular cartilage extracellular matrix (dECM), dECM can be derived from porcine cartilage, bovine cartilage, rabbit cartilage, sheep cartilage and so on.
  • acellular cartilage extracellular matrix can be prepared by physical decellularization methods known in the art (for example, freeze-thaw method), and then chemical methods (for example, using a hydrogen peroxide solution with a mass concentration of 3%) can be used to treat the extracellular matrix of the acellular cartilage cells.
  • the treatment of the matrix can be further decellularized and sterilized, so that the acellular cartilage extracellular matrix has lower immunogenicity, improves the biocompatibility of the cartilage composite scaffold, and reduces the receptor's immune rejection of the implanted cartilage composite scaffold .
  • the compressive elastic modulus of the tissue engineering cartilage composite scaffold is 100 MPa to 600 MPa, and further is 200 MPa to 500 MPa.
  • the tissue engineering cartilage composite scaffold exhibits high mechanical properties and can fully meet the requirements of mechanical support.
  • the fourth aspect of the embodiments of the present application provides a method for preparing a tissue engineered cartilage composite scaffold, by which the above-mentioned tissue engineered cartilage composite scaffold can be prepared.
  • the preparation method includes the following steps:
  • the matrix material and the aforementioned growth factor sustained-release microspheres can be dissolved in deionized water to prepare a first aqueous solution.
  • concentration of the matrix material in the first aqueous solution is preferably 0.01 g/mL to 0.05 g/mL.
  • concentration of the growth factor sustained-release microspheres in the first aqueous solution is preferably 0.01 g/mL to 0.05 g/mL.
  • a hydrogel such as GelMA is dissolved in deionized water to form a second aqueous solution with a concentration of the hydrogel ranging from 0.1 g/mL to 0.2 g/mL.
  • the mixing ratio of the first aqueous solution and the second aqueous solution is preferably 1:1 to 1:2.
  • the mixing ratio of the first aqueous solution and the second aqueous solution is within the above range, and the obtained hydrogel fiber can be more bionic extracellular matrix and provide an excellent microenvironment for cell growth.
  • the bio-ink is used as the raw material of the hydrogel fiber
  • the degradable polymer material is used as the raw material of the degradable polymer fiber
  • the tissue engineering cartilage composite scaffold is obtained by printing.
  • step S400 printing can be performed in a bioprinter.
  • the ALPHA-IPT1 biological 3D printer of Shangpu Company can separate the biomedicine and the degradable polymer material into two barrels of the printer, each of the two barrels It is connected with a printing head and controls the two printing heads to perform printing and molding according to a preset printing path, and then undergo cross-linking treatment to obtain a tissue engineering cartilage composite scaffold.
  • the cross-linking treatment can improve the mechanical properties of the tissue engineering cartilage composite scaffold.
  • the cross-linking treatment of the matrix material can also improve the degradation rate of the matrix material, prevent its shrinkage and deformation, ensure the appearance and shape of the tissue engineering cartilage composite scaffold and its internal specific microstructure, which is conducive to cell growth, proliferation and redifferentiation.
  • step S400 may include:
  • the diameter range of the first print head and the second print head are 200 ⁇ m ⁇ 800 ⁇ m, the extrusion speed can be 0.01mm/s ⁇ 0.05mm/s, the printing speed can be 5mm/s ⁇ 15mm/s, and the layer thickness range It can be 0.1mm ⁇ 0.5mm.
  • step S440 one or more of chemical methods, irradiation methods, and dry heat methods known in the art may be used to cross-link the printed product.
  • an irradiation method can be used to cross-link the printed product to obtain a tissue engineered cartilage composite scaffold.
  • the irradiation method is, for example, electron beam irradiation, ultraviolet light irradiation, and gamma ray irradiation.
  • Radiation cross-linking treatment can reduce the amount of cross-linking agent used, or not use cross-linking agent, which can improve the biocompatibility of tissue engineering cartilage composite scaffold.
  • the cross-linking agent can be carbodiimide (EDAC), N-hydroxysuccinimide (NHS), LAP photocross-linking agent, Genipin and glutaraldehyde (GDA) one or more.
  • EDAC carbodiimide
  • NHS N-hydroxysuccinimide
  • GDA glutaraldehyde
  • the concentration of the crosslinking agent in the first aqueous solution may be 100 ⁇ g/mL to 200 ⁇ g/mL.
  • the w 1 /o/w 2 emulsification-solvent volatilization method was used to prepare PLGA containing rhTGF- ⁇ 3 growth factor sustained-release microspheres.
  • the specific process is as follows: 2 ⁇ g of rhTGF- ⁇ 3 was dissolved in 200 ⁇ L of deionized water to prepare a concentration of 10 ⁇ g/mL The rhTGF- ⁇ 3 aqueous solution is used as the internal water phase w 1 .
  • a polylactic acid-glycolic acid copolymer (PLGA) with a molecular weight of 102,000 Da was dissolved in dichloromethane (DCM) to prepare a PLGA solution with a concentration of 25 mg/mL as the oil phase o.
  • DCM dichloromethane
  • PVA polyvinyl alcohol
  • aqueous PVA solution with a mass concentration of 1% as the outer water phase w 2 ; add 200 ⁇ L of the inner water phase w 1 to the 2 mL oil phase o
  • use the American Qsonica Q125 ultrasonic breaker probe to shake at 60% amplitude (ultrasonic power is 75w) for 30 seconds to form a primary emulsion (w 1 /o); then add the primary emulsion (w 1 /o) to 20 mL of external water In phase w 2 , at room temperature (20°C ⁇ 25°C), magnetically stirred at 500 rpm for 12 hours to evaporate and remove DCM to obtain a microsphere solution; then centrifuge the microsphere solution at 2000 rpm for 3 minutes and discard the supernatant to obtain the initial microsphere solution.
  • PVA polyvinyl alcohol
  • the balls were washed three times with deionized water, and then freeze-dried at -20°C for 24 hours to obtain pure growth factor sustained-release microspheres with a particle size of 10 ⁇ m-100 ⁇ m, which were stored at -20°C for later use.
  • Example 1 The difference from Example 1 is that 5 ⁇ g of rhTGF- ⁇ 3 was dissolved in 200 ⁇ L of deionized water to prepare an aqueous solution of rhTGF- ⁇ 3 with a concentration of 25 ⁇ g/mL as the internal water phase w 1 .
  • Example 2 The difference from Example 1 is that 10 ⁇ g of rhTGF- ⁇ 3 was dissolved in 200 ⁇ L of deionized water to prepare an aqueous solution of rhTGF- ⁇ 3 with a concentration of 50 ⁇ g/mL as the internal water phase w 1 .
  • Figure 1 show that the growth factor sustained-release microspheres of Example 1 are spherical and well-shaped, with a diameter of 5 ⁇ m-30 ⁇ m, a smooth surface with slight undulations, and the distance between two adjacent wave crests is 0.38 ⁇ m ⁇ 0.71 ⁇ m.
  • the growth factor sustained-release microspheres of the examples of the present application have a good microsphere appearance, which is conducive to cell adhesion and promotes cell growth, migration, proliferation and redifferentiation.
  • the supernatants after centrifugation of the microsphere solutions in Examples 1-3 were collected, and the optical density (OD) value of the supernatants was determined by using enzyme-linked immunosorbent assay (Enzyme-Linked ImmunoSorbent Assay, referred to as ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the standard curve equation was used to calculate the concentration and quality of rhTGF- ⁇ 3 in the supernatant, and the following formula was used to calculate the encapsulation amount and encapsulation efficiency of rhTGF- ⁇ 3 in the growth factor sustained-release microspheres. A total of 3 measurements were made and the results were averaged.
  • Encapsulation rate (the mass of rhTGF- ⁇ 3 in the microsphere/the total mass of rhTGF- ⁇ 3) ⁇ 100%
  • Encapsulation amount mass of rhTGF- ⁇ 3 in microsphere/mass of microsphere, where the unit of encapsulation amount is ng/mg, the unit of rhTGF- ⁇ 3 in microsphere is ng, and the unit of microsphere mass is mg.
  • the mass of rhTGF- ⁇ 3 in the microspheres the total mass of rhTGF- ⁇ 3-the mass of rhTGF- ⁇ 3 in the supernatant
  • Example 1 the average encapsulation efficiency of rhTGF- ⁇ 3 in the growth factor sustained-release microspheres was 93.6%, and the average encapsulation amount was 37.5 ng/mg.
  • Example 2 the average encapsulation efficiency of rhTGF- ⁇ 3 in the growth factor sustained-release microspheres was 94.4%, and the average encapsulation amount was 94.4 ng/mg.
  • Example 3 the average encapsulation efficiency of rhTGF- ⁇ 3 in the growth factor sustained-release microspheres was 98.9%, and the average encapsulation amount was 197.8 ng/mg.
  • PLGA has a high encapsulation rate of rhTGF- ⁇ 3 and an effective encapsulation amount, which can improve the utilization rate of rhTGF- ⁇ 3.
  • a certain quality of growth factor sustained-release microspheres was added to 1 mL of phosphate buffered saline solution PBS (pH 7.4), and then placed on a shaker at room temperature (20°C ⁇ 25°C) with a rotation speed of 80 rpm. After sustained release for a certain period of time, centrifuge at 10000rcf for 5 minutes, take the supernatant and save, and add the same amount of fresh PBS buffer (pH 7.4) to continue sustained release; take multiple supernatant samples in this way during the sustained release cycle .
  • An ELISA kit was used to determine the concentration of rhTGF- ⁇ 3 in the supernatant sample and calculate the cumulative release of rhTGF- ⁇ 3. Each group tests 3 samples, and the results are averaged.
  • Example 2 Addition of growth factor sustained-release microspheres (mg) 24.2 8.3 4.1 Sustained release amount on the first day (%) 0.052 0.088 0.338 Sustained release amount in the first week (%) 0.135 0.228 0.704
  • the sustained release amounts of Examples 1 to 3 are all based on the initial rhTGF- ⁇ 3 content of the microspheres.
  • the growth factor sustained-release microspheres of Examples 1 to 3 can achieve long-term and effective controlled-release of rhTGF- ⁇ 3, and have the maximum sustained-release rate on the first day, and basically reach a stable sustained-release rate after the first week.
  • the sustained-release period of the growth factor sustained-release microspheres of Examples 1 to 3 can reach more than 500 days, which can realize the long-term and gradual slow-release of rhTGF- ⁇ 3 in the microspheres, thereby exerting the cell-recruiting ability of rhTGF- ⁇ 3 for a long time and satisfying the requirement of cartilage defect. Long-term regeneration and repair needs of the organization.
  • Figure 4 shows the 28-day cumulative in vitro release curve of the growth factor sustained-release microspheres of Examples 1 to 3, including the cumulative release of sustained-release time of 1, 2, 4, 7, 14, 21, and 28 days. degree.
  • growth factor sustained-release microspheres with different encapsulated amounts of rhTGF- ⁇ 3 have different sustained-release speeds.
  • the release rate of microspheres with relatively high rhTGF- ⁇ 3 encapsulation is also relatively high.
  • the growth factor sustained-release microspheres with different encapsulated amounts of rhTGF- ⁇ 3 did not have a convex release phenomenon.
  • the test results show that the growth factor sustained-release microspheres rhTGF- ⁇ 3 in the examples of this application have a slow release rate, and are a good controlled-release carrier, achieving slow release of rhTGF- ⁇ 3, continuous recruitment of stem cells and promotion of stem cell chondrocyte differentiation.
  • the purpose is to provide a good treatment effect for cartilage defects that require long-term growth and recovery.
  • the experiment set up 6 experimental groups:
  • Negative group DMEM/F12 (50:50) low sugar medium, which contains 1% fetal bovine serum FBS.
  • DMEM/F12 (50:50) low-sugar medium is a 1:1 combination of F12 medium (Ham's F 12 nutrient medium animal cell medium) and DMEM (Dulbecco's Modified Eagle Medium) medium.
  • Blank group DMEM/F12 (50:50) low sugar medium containing 1% FBS and 10 mg/mL blank PLGA microspheres.
  • Experimental group 1 DMEM/F12 (50:50) low-sugar medium, which contains 1% FBS and 5 mg/mL of the growth factor sustained-release microspheres of Example 3.
  • the content of the growth factor sustained-release microspheres meets rhTGF-
  • the content of factor ⁇ 3 is about 1000ng/mL medium.
  • Experimental group 2 DMEM/F12 (50:50) low-sugar medium containing 1% FBS and 0.5 mg/mL of the growth factor sustained-release microspheres of Example 3.
  • the content of the growth factor sustained-release microspheres meets rhTGF
  • the content of - ⁇ 3 factor is about 100ng/mL medium.
  • Experimental group 3 DMEM/F12 (50:50) low-sugar medium containing 1% FBS and 0.05 mg/mL of the growth factor sustained-release microspheres of Example 3.
  • the content of the growth factor sustained-release microspheres meets rhTGF
  • the content of - ⁇ 3 factor is about 10ng/mL medium.
  • the dECM source taken from rabbit cartilage was first treated by freezing and thawing, and then treated with a hydrogen peroxide solution with a mass concentration of 3% to obtain the acellular cartilage extracellular matrix; the acellular cartilage extracellular matrix was grown as in Example 1.
  • the factor sustained-release microspheres and the LAP photocrosslinker are dissolved in deionized water to form the first aqueous solution with the concentration of both the acellular cartilage extracellular matrix and the growth factor sustained-release microspheres of 0.03g/mL.
  • the concentration of the coupling agent is 100 ⁇ g/mL; the GelMA hydrogel is dissolved in deionized water to prepare a second aqueous solution with a concentration of 0.2g/mL of GelMA hydrogel, in which the degree of methylation of GelMA hydrogel is about 60 %; The first aqueous solution and the second aqueous solution are mixed in a volume ratio of 1:1 to obtain a biological ink with printing performance.
  • Adopt the ALPHA-IPT1 biological 3D printer of Shangpu Company add the biological ink into the cylinder connected to the first print head of the bioprinter, and set the heat preservation temperature to 16°C; add polycaprolactone particles to the second and second biological printer In the barrel connected to the print head, set the heat preservation temperature to 100°C; adjust the temperature of the bioprinter molding room to 5°C; according to the structure of Fig. 8 and Fig. 9, the first print head and the second print head are parallel according to the preset printing path After printing, the printed product is cross-linked by ultraviolet light to obtain a tissue engineering cartilage composite scaffold.
  • the growth factor sustained-release microspheres are the growth factor sustained-release microspheres of Example 2.
  • the growth factor sustained-release microspheres are the growth factor sustained-release microspheres of Example 3.
  • the general view of the tissue engineering cartilage composite scaffold in Example 4 is shown in FIG. 7, and the microscopic view is shown in FIG. 10.
  • the results in Figure 7 show that the tissue engineering cartilage composite scaffold is a 20mm ⁇ 20mm ⁇ 2mm rectangular parallelepiped frame structure, the PCL fibers are white, the hydrogel fibers are transparent, and there are small bubbles in the pores.
  • the results in Figure 10 show that the width of the PCL fibers of the tissue engineering cartilage composite scaffold is about 700 ⁇ m, the average pores between the fiber groups are 500 ⁇ m, and the width of the hydrogel fibers is about 500 ⁇ m.
  • Example 4 Compression modulus of elasticity/MPa 400 400 500
  • the compressive elastic modulus of the tissue engineering cartilage composite scaffold of the examples of the application can reach 400 MPa to 500 MPa, which is higher than the compressive elastic modulus of normal cartilage, and can provide better mechanical support.
  • the sterile tissue engineering cartilage composite scaffold prepared in Example 4 was co-cultured with New Zealand rabbit second-generation adipose-derived mesenchymal stem cells for 7 days, then stained with a live/dead cell staining kit, and photographed with a fluorescent confocal microscope to evaluate tissue engineering The biocompatibility of cartilage composite scaffolds. The result is shown in Figure 11.
  • the cells marked by the closed curve are dead cells, and the rest are live cells, and the cell survival rate can reach about 95%.
  • the results in Figure 11 show that the cells grow well in the tissue engineered cartilage composite scaffold, and the cell survival rate is high, indicating that the tissue engineered cartilage composite scaffold of the examples of the present application has good biocompatibility and is beneficial to cell survival, proliferation and redifferentiation. .
  • the sterile tissue engineered cartilage composite scaffold prepared in Example 4 was co-cultured with mouse RAW cells for 3 days, and then fixed with 2.5% glutaraldehyde. After pretreatment, the tissue engineered cartilage composite scaffold was observed under a scanning electron microscope to evaluate the biological characteristics of the tissue engineering cartilage composite scaffold. compatibility. The result is shown in Figure 12.
  • tissue engineered cartilage composite scaffold provided by the examples of the application has a porous structure, good biocompatibility, and high mechanical strength. It is suitable for the transplantation of cartilage defects and can well promote the cartilage defect in the avascular area. Regenerative repair to improve the regeneration effect.

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Abstract

一种生长因子缓释微球、组织工程软骨复合支架及制备方法。生长因子缓释微球包括聚乳酸-羟基乙酸共聚物基质材料以及包封于基质材料中的重组人转化生长因子β3；微球中重组人转化生长因子β3的包封量为5ng/mg～200ng/mg；微球的缓释期为28天以上。

Description

生长因子缓释微球、组织工程软骨复合支架及制备方法
相关申请的交叉引用
本申请要求享有于2019年05月20日提交的名称为“生长因子缓释微球、组织工程软骨复合支架及制备方法”的中国专利申请201910417734.2的优先权，该申请的全部内容通过引用并入本文中。
技术领域
本申请属于医用材料技术领域，具体涉及一种生长因子缓释微球、组织工程软骨复合支架及制备方法。
背景技术
软骨是人或动物体内骨骼系统的重要组成部分，软骨组织为高度特化的结缔组织，其特点是组织中缺乏血管、神经及淋巴管，细胞外基质呈致密的固态，软骨细胞被大量的细胞外基质(如胶原、纤维、蛋白、多糖等)包绕。正是由于软骨的组织学特性、生化成分的构成及软骨细胞处于低代谢和低增殖状态的特征，限制了软骨细胞的增生反应及向损伤区域的迁移，导致损伤后的软骨组织进行自我修复与再生的能力较低，难以实现自身修复及再生，并会逐步加重，导致骨关节炎，引起关节功能障碍。
发明内容
第一方面，本申请提供一种生长因子缓释微球，其包括聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid)，简称PLGA)基质材料以及包封于基质材料中的重组人转化生长因子β3(Recombinant Human Transforming Growth Factor-beta 3，简称rhTGF-β3)；微球中rhTGF-β3的包封量为5ng/mg～200ng/mg；微球的缓释期为28天以上。
第二方面，本申请提供一种生长因子缓释微球的制备方法，其包括以 下步骤：
提供浓度为1μg/mL～100μg/mL的rhTGF-β3水溶液；
提供浓度为20mg/mL～25mg/mL的PLGA油相溶液；
提供浓度为5mg/mL～10mg/mL的聚乙烯醇(polyvinyl alcohol，简称PVA)水溶液；
将rhTGF-β3水溶液与PLGA油相溶液在冰浴中超声混合，得到初级乳液；
将初级乳液与PVA水溶液在20℃～25℃下搅拌混合，得到微球溶液；
将微球溶液分离处理，得到初始微球经洗涤、冷冻干燥后，得到生长因子缓释微球，微球中rhTGF-β3的包封量为5ng/mg～200ng/mg，微球的缓释期为28天以上。
第三方面，本申请提供一种组织工程软骨复合支架，其包括：
组织工程支架，包括沿自身高度方向依次层叠的多个支架层，相邻两个支架层中的一者包括多个沿第一方向相互间隔设置的纤维组，另一者包括多个沿第二方向相互间隔设置的纤维组，每个纤维组包括可降解高分子纤维及设置于可降解高分子纤维自身宽度方向的一侧或两侧的水凝胶纤维，水凝胶纤维沿可降解高分子纤维的长度方向延伸，第一方向与第二方向相交；
基质材料，复合于组织工程支架的水凝胶纤维中；
上述的生长因子缓释微球，复合于组织工程支架的水凝胶纤维中。
第四方面，本申请提供一种组织工程软骨复合支架的制备方法，其包括以下步骤：
提供包含基质材料和所述生长因子缓释微球的第一水溶液；
提供包含水凝胶的第二水溶液；
将第一水溶液和第二水溶液混合，得到生物墨水；
以生物墨水作为水凝胶纤维的原料、以可降解高分子材料作为可降解高分子纤维的原料，打印得到上述的组织工程软骨复合支架。
相对于现有技术，本申请至少具有以下有益效果：
本申请提供的生长因子缓释微球、组织工程软骨复合支架及制备方 法，其中生长因子缓释微球具有rhTGF-β3控缓释放性能好的特点，能够实现长期有效的rhTGF-β3释放浓度，以及长期有效的发挥rhTGF-β3的细胞募集能力，达到提高缺损软骨组织的再生修复效果。
进一步地，组织工程软骨复合支架很好地仿生了软骨胶原取向和成分分布以及细胞生长的微环境，融合了可降解高分子纤维良好的力学性能和生物可降解性、基质材料良好的生物相容性、以及生长因子缓释微球的可控缓释性能和强大的细胞募集能力，有利于细胞的生长、迁移、增殖及再分化，从而可以很好地促进无血管区的部分或全部缺损软骨组织的再生修复，使新生软骨组织具有优良的形态、力学性能及生理功能。
附图说明
为了更清楚地说明本申请实施例的技术方案，下面将对本申请实施例中所需要使用的附图作简单地介绍，显而易见地，下面所描述的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据附图获得其他的附图。
图1为本申请实施例1的生长因子缓释微球的扫描电子显微镜(SEM)图像。
图2为本申请实施例2的生长因子缓释微球的扫描电子显微镜(SEM)图像。
图3为本申请实施例3的生长因子缓释微球的扫描电子显微镜(SEM)图像。
图4为本申请实施例1至3的生长因子缓释微球体外累计释放度曲线图。
图5为Transwell迁移实验结果图像，其中A为阴性组；B为阳性组；C为空白组；D为实验组1；E为实验组2；F为实验组3。
图6为Transwell迁移实验结果统计分析图，其中A为阴性组；B为阳性组；C为空白组；D为实验组1；E为实验组2；F为实验组3。
图7为本申请实施例的组织工程软骨复合支架的结构图。
图8为本申请实施例的组织工程软骨复合支架的截面示意图。
图9为本申请实施例的组织工程软骨复合支架中支架层的示意图。
图10为本申请实施例的组织工程软骨复合支架的普通光学显微镜镜图像。
图11为本申请实施例的组织工程软骨复合支架负荷兔脂肪干细胞共聚焦显微镜图像。
图12为本申请实施例的组织工程软骨复合支架负荷小鼠巨噬RAW细胞SEM图像。
具体实施方式
为了使本申请的发明目的、技术方案和有益技术效果更加清晰，以下结合具体实施例对本申请进行详细说明。应当理解的是，本说明书中描述的实施例仅仅是为了解释本申请，并非为了限定本申请。
为了简便，本文仅明确地公开了一些数值范围。然而，任意下限可以与任何上限组合形成未明确记载的范围；以及任意下限可以与其它下限组合形成未明确记载的范围，同样任意上限可以与任意其它上限组合形成未明确记载的范围。此外，尽管未明确记载，但是范围端点间的每个点或单个数值都包含在该范围内。因而，每个点或单个数值可以作为自身的下限或上限与任意其它点或单个数值组合或与其它下限或上限组合形成未明确记载的范围。
在本文的描述中，需要说明的是，除非另有说明，“以上”、“以下”为包含本数，“一种或多种”中“多种”的含义是两种以上。
本申请的上述发明内容并不意欲描述本申请中的每个公开的实施方式或每种实现方式。如下描述更具体地举例说明示例性实施方式。在整篇申请中的多处，通过一系列实施例提供了指导，这些实施例可以以各种组合形式使用。在各个实例中，列举仅作为代表性组，不应解释为穷举。
组织工程学是综合应用工程学和生命科学的原理与技术，在体外预先构建一个有生物活性的种植体，然后植入体内，达到修复组织缺损和重建组织功能的目的。为了实现将组织工程骨应用于临床软骨缺损的治疗，本申请实施例提供一种生长因子缓释微球、组织工程软骨复合支架及制备方 法。
首先说明本申请第一方面的实施例提供的一种生长因子缓释微球，微球包括PLGA基质材料以及包封于基质材料中的rhTGF-β3；微球中rhTGF-β3的包封量为5ng/mg～200ng/mg；微球的缓释期为28天以上。
本申请实施例提供的生长因子缓释微球具有rhTGF-β3控缓释放性能好的特点，能够实现长期有效的rhTGF-β3释放浓度，以及长期有效的发挥rhTGF-β3的细胞募集能力，达到提高缺损软骨组织的再生修复效果。
采用PLGA对rhTGF-β3进行包封，实现rhTGF-β3的长期缓释，避免了rhTGF-β3作用时间短、易被降解和作用一过性等问题，有效防止rhTGF-β3因新陈代谢过程、生理环境变化、或与酶的作用而变性，更好地发挥rhTGF-β3的生物学作用。
本申请实施例提供的生长因子缓释微球能够应用中组织工程软骨复合支架中，达到对缺损软骨组织长期有效的再生修复效果。
在一些实施例中，生长因子缓释微球中rhTGF-β3的包封量为30ng/mg～200ng/mg。
进一步地，微球在第一天的缓释量为0.05％～0.5％，微球在第一天具有最大缓释速率。让rhTGF-β3以较小的速率逐渐缓释，既能达到长期的局部较小有效药物刺激浓度，又能抑制rhTGF-β3过量的潜在并发症，对于需要长时间生长和恢复的软骨缺陷来说提供良好的治疗效果，并且提高成骨质量、降低炎症反应及其他风险。
进一步地，微球在第一周的缓释量为0.1％～1％。
上述的“第一天的缓释量”及“第一周的缓释量”均是以微球的初始rhTGF-β3含量为基准计算的。
在一些实施例中，生长因子缓释微球的缓释期可以达到半年以上，如200天～600天，再例如300天～600天。
在一些实施例中，PLGA的分子量优选为1×10 5Da～1.1×10 5Da，更优选为1.02×10 5Da。
在一些实施例中，PLGA中乳酸与羟基乙酸的摩尔比优选为1:1～5:1，例如2.5:1～3.5:1，再例如为3:1。
在一些实施例中，生长因子缓释微球的粒径优选为5μm～100μm，更优选为5μm～50μm。这有利于更好地实现rhTGF-β3的控缓释放。
在一些实施例中，生长因子缓释微球的表面可以具有轻微起伏，相邻两波峰之间的间距例如为0.1μm～2.5μm，进一步为0.3μm～2μm。生长因子缓释微球微球形貌良好，有利于细胞粘附，促进细胞的生长、迁移、增殖及再分化，提高缺损软骨组织的再生修复效果。
本申请实施例第二方面提供一种生长因子缓释微球的制备方法，通过该制备方法能够制备得到上述的生长因子缓释微球。制备方法包括以下步骤：
S10，提供浓度为1μg/mL～100μg/mL的rhTGF-β3水溶液。
在步骤S10，可以是将rhTGF-β3溶于去离子水中，制备浓度为1μg/mL～100μg/mL的rhTGF-β3水溶液，作为内水相w 1。
S20，提供浓度为20mg/mL～25mg/mL的PLGA油相溶液。
在步骤S20，可以是将PLGA溶于二氯甲烷(DCM)中，制备浓度为20mg/mL～25mg/mL的PLGA油相溶液，作为油相o。该种油相o能够对rhTGF-β3具有较高的包封率。
在步骤S20，PLGA的分子量优选为1×10 5Da～1.1×10 5Da，更优选为1.02×10 5Da。进一步地，PLGA中乳酸与羟基乙酸的摩尔比优选为1:1～5:1，例如2.5:1～3.5:1，再例如为3:1。rhTGF-β3与该些PLGA之间的作用较好，能够提高生长因子缓释微球对rhTGF-β3的包封率，并使得生长因子缓释微球具有改善的rhTGF-β3控缓释放速率，延长缓释周期，满足软骨缺损的长期再生修复需求。并且，该些PLGA的生物可降解性较好，能够为细胞附着、增殖和分化提供优良载体。
S30，提供浓度为5mg/mL～10mg/mL的PVA水溶液。
在步骤S30，将PVA溶于去离子水中，制备浓度为5mg/mL～10mg/mL的PVA水溶液，作为外水相w 2。该外水相w 2能够为生长因子缓释微球的形成提供优良的环境。
在步骤S30，PVA水溶液中聚乙烯醇的分子量优选为6×10 4Da～7×10 4Da，更优选为6.7×10 4Da。
S40，将rhTGF-β3水溶液与PLGA油相溶液在冰浴中超声混合，得到初级乳液。
在步骤S40，通过将rhTGF-β3水溶液与PLGA油相溶液在冰浴中超声混合，能够将其充分均质乳化，有利于提高PLGA对rhTGF-β3的包封率，以及提高rhTGF-β3在微球中的分散均匀性，从而提高rhTGF-β3的利用率，并改善rhTGF-β3的控缓释放效果。此外，还有效保存了rhTGF-β3的生物学活性。
在步骤S40，rhTGF-β3水溶液与PLGA油相溶液混合的体积比优选为1:5～1:10。
在步骤S40，超声混合的超声功率优选为70W～75W，超声时间优选为10s～30s。
步骤S40可以在超声细胞粉碎机中进行，如美国Qsonica Q125型超声波破碎仪，使破碎仪探头以60％振幅(超声功率为75W)震荡10s～30s，将rhTGF-β3水溶液与PLGA油相溶液超声混合，得到初级乳液。
S50，将初级乳液与PVA水溶液在20℃～25℃下搅拌混合，得到微球溶液。
在步骤S50，rhTGF-β3与PLGA自组装成微球，搅拌过程中还使得DCM挥发除去。
在步骤S50，初级乳液与PVA水溶液混合的体积比可以为1:5～1:25，例如为1:8～1:12。
进一步地，搅拌混合的速率优选为500rpm～1000rpm，时间优选为4h～12h。
S60，将微球溶液分离处理，得到初始微球经洗涤、冷冻干燥后，得到上述的生长因子缓释微球。
在步骤S60，可以采用抽滤法、离心分离法等方法将微球溶液分离处理得到初始微球。例如，将微球溶液在1500rpm～2000rpm下离心分离3min～5min。
可以采用去离子水对初始微球进行反复清洗2～4次，如3次。
可以将经洗涤后的初始微球在-4～-20℃下冷冻干燥18h～24h，得到生 长因子缓释微球。
将制备得到的生长因子缓释微球于-4℃～-25℃的条件下保存备用，例如在-20℃的条件下保存备用。
采用本申请实施例的生长因子缓释微球的制备方法，对rhTGF-β3还具有较高的包封率，可以达到80％以上，进一步地90％以上，能够提高rhTGF-β3的利用率。
接下来说明本申请实施例第三方面提供的一种组织工程软骨复合支架，请参照图7至图9，组织工程软骨复合支架包括组织工程支架以及复合于组织工程支架的基质材料和上述的生长因子缓释微球。
组织工程支架呈多孔框架结构体，其包括沿自身高度方向依次层叠的多个支架层1，相邻两个支架层1中的一者包括多个沿第一方向(如图8中的X方向)相互间隔设置的纤维组2，另一者包括多个沿第二方向(如图8中垂直于纸面的方向)相互间隔设置的纤维组2，每个纤维组2包括可降解高分子纤维3及设置于可降解高分子纤维自身宽度方向的一侧或两侧的水凝胶纤维4，水凝胶纤维沿可降解高分子纤维的长度方向延伸，其中第一方向与第二方向相交，优选地，第一方向与第二方向垂直。
基质材料和生长因子缓释微球均复合于组织工程支架的水凝胶纤维4中。
本申请实施例提供的组织工程软骨复合支架很好地仿生了软骨胶原取向和成分分布以及细胞生长的微环境，融合了可降解高分子纤维3良好的力学性能和生物可降解性、基质材料良好的生物相容性、以及生长因子缓释微球的可控缓释性能和强大的细胞募集能力，使得复合支架具有良好的生物相容性、力学性能、可降解性能、较低的免疫原性和生长因子缓释性，并可以通过募集内源性干细胞再生修复缺损软骨组织，促进细胞的生长、迁移、增殖及再分化，从而很好地促进无血管区的部分或全部缺损软骨组织的再生修复，使新生软骨组织具有优良的形态、力学性能及生理功能。
请一并参照图10，在支架层1中，相邻纤维组2之间的间隔优选为300μm～500μm；相邻纤维组2中可降解高分子纤维3之间的间隔优选为 750μm～1500μm；支架层1的高度优选为100μm～500μm。该种组织工程软骨复合支架更好地仿生了软骨组织的取向性大孔结构，以及在大孔结构内部的小孔结构，使得组织工程软骨复合支架具有较高的力学性能及生物相容性的同时，还能够让内源性干细胞更易于迁移，并在迁移过程中完成增殖和分化，实现部分软骨缺损或全部软骨缺损的再生修复，具有较高的修复效果。
进一步地，可降解高分子纤维的宽度优选为200μm～800μm；水凝胶纤维的宽度优选为200μm～800μm。
可降解高分子纤维的材料可以选自聚己内酯PCL、聚氨酯PU、聚乳酸PLA、聚乳酸-羟基乙酸共聚物PLGA、聚乳酸-聚己内酯共聚物PCLA、聚氨基酸PAA及聚乙醇酸PGA中的一种或多种。例如为PCL，其具有良好的力学性能和可生物降解性。进一步地，PCL的分子量优选为2×10 4Da～5×10 5Da，如2×10 4Da～1×10 5Da。
在一些实施例中，水凝胶纤维优选为甲基化程度为40％～80％的甲基丙烯酸化水凝胶(GelMA)纤维，进一步优选为甲基化程度为50％～70％的GelMA纤维。该种水凝胶纤维具有良好的可打印性。另外，采用该种水凝胶纤维有利于细胞的附着、迁移、增殖和分化。
基质材料可以选自脱细胞软骨细胞外基质、I型胶原、II型胶原、细菌纤维素、蚕丝蛋白及糖胺多糖中的一种或多种，例如为脱细胞软骨细胞外基质(dECM)，dECM可来源于猪软骨、牛软骨、兔软骨、羊软骨等。可通过本领域已知的物理脱细胞的方法(例如冻融法)制备脱细胞软骨细胞外基质，之后采用化学法(例如使用质量浓度为3％的过氧化氢溶液)对脱细胞软骨细胞外基质进行处理，可以进一步脱细胞及灭菌，使得脱细胞软骨细胞外基质具有较低的免疫原性，提高软骨复合支架的生物相容性，降低受体对植入软骨复合支架的免疫排斥作用。
在一些实施例中，组织工程软骨复合支架的压缩弹性模量为100MPa～600MPa，进一步地为200MPa～500MPa。该组织工程软骨复合支架表现出较高的力学性能，完全能够满足力学支撑的需求。
本申请实施例第四方面提供一种组织工程软骨复合支架的制备方法， 通过该制备方法能够制备得到上述的组织工程软骨复合支架。制备方法包括以下步骤：
S100，提供包含基质材料和上述生长因子缓释微球的第一水溶液。
在步骤S100，可以将基质材料和上述生长因子缓释微球溶于去离子水中制成第一水溶液。进一步地，第一水溶液中基质材料的浓度优选为0.01g/mL～0.05g/mL。第一水溶液中生长因子缓释微球的浓度优选为0.01g/mL～0.05g/mL。
S200，提供包含水凝胶的第二水溶液。
在步骤S200，将水凝胶如GelMA溶于去离子水中，制成水凝胶的浓度为0.1g/mL～0.2g/mL的第二水溶液。
S300，将第一水溶液和第二水溶液混合，得到具有打印性能的生物墨水。
在步骤S300，第一水溶液与第二水溶液的混合比例优选为1:1～1:2。第一水溶液与第二水溶液的混合比例在上述范围内，得到的水凝胶纤维能够更仿生细胞外基质，为细胞生长提供优良的微环境。
S400，以生物墨水作为水凝胶纤维的原料、以可降解高分子材料作为可降解高分子纤维的原料，打印得到组织工程软骨复合支架。
在步骤S400，打印可以在生物打印机中进行，如上普公司的ALPHA-IPT1型生物3D打印机，可以将生物药水和可降解高分子材料分装于打印机的两个料筒中，该两个料筒各自与一打印头连接，控制两个打印头按照预设的打印路径进行打印成型，之后经交联处理，得到组织工程软骨复合支架。
通过交联处理，能够提高组织工程软骨复合支架的力学性能。其中基质材料交联处理还能够改善基质材料的降解速率，防止其收缩变形，保证组织工程软骨复合支架的外观形态及其内部的特定微结构，有利于细胞的生长、增殖及再分化。
作为示例，步骤S400可以包括：
S410，将生物墨水加入生物打印机的与第一打印头相连的料筒中，设置保温温度为12℃～16℃。
S420，将聚己内酯加入生物打印机的与第二打印头相连的料筒中，设置保温温度为80℃～100℃。
S430，将生物打印机成型室的温度调整为3℃～5℃。
S440，第一打印头与第二打印头并行进行打印，打印产品经交联处理得到组织工程软骨复合支架。
第一打印头与第二打印头的直径范围分别为200μm～800μm，挤出速度范围可以为0.01mm/s～0.05mm/s，打印速度范围可以为5mm/s～15mm/s，层厚范围可以为0.1mm～0.5mm。
在步骤S440，可以采用本领域已知的化学方法、辐照方法及干热方法中的一种或多种对打印产品进行交联处理。
作为示例，可以采用辐照方法对打印产品进行交联处理，得到组织工程软骨复合支架。辐照方法例如是电子束辐照、紫外光辐照、γ射线辐照。辐照交联处理可以减少交联剂的使用量，或者不使用交联剂，这能够提高组织工程软骨复合支架的生物相容性。
当交联处理使用交联剂时，交联剂可以是碳二亚胺(EDAC)、N-羟基琥珀酰亚胺(NHS)、LAP光交联剂、京尼平(Genipin)及戊二醛(GDA)中的一种或多种。例如，将交联剂预先加入第一水溶液中，第一水溶液中交联剂的浓度可以为100μg/mL～200μg/mL。
实施例
下述实施例更具体地描述了本申请公开的内容，这些实施例仅仅用于阐述性说明，因为在本申请公开内容的范围内进行各种修改和变化对本领域技术人员来说是明显的。除非另有声明，以下实施例中所报道的所有份、百分比、和比值都是基于重量计，而且实施例中使用的所有试剂都可商购获得或是按照常规方法进行合成获得，并且可直接使用而无需进一步处理，以及实施例中使用的仪器均可商购获得。
实施例1
采用w 1/o/w 2乳化-溶剂挥发法制备PLGA载rhTGF-β3的生长因子缓释微球，具体过程如下：将2μg的rhTGF-β3溶于200μL去离子水中，制备浓 度为10μg/mL的rhTGF-β3水溶液，作为内水相w 1。将分子量为10.2万Da的聚乳酸-羟基乙酸共聚物(PLGA)溶于二氯甲烷(DCM)中，制备浓度为25mg/mL的PLGA溶液，作为油相o。将分子量为6.7万Da的聚乙烯醇(PVA)溶于去离子水中，制备质量浓度为1％的PVA水溶液，作为外水相w 2；将200μL内水相w 1加入到2mL油相o中，冰浴中用美国Qsonica Q125型超声波破碎仪探头以60％振幅(超声功率为75w)震荡30秒形成初级乳液(w 1/o)；再将初级乳液(w 1/o)加入20mL外水相w 2中，在室温(20℃～25℃)下，以500rpm磁力搅拌12小时，挥发除去DCM，得到微球溶液；再将微球溶液以2000rpm离心3分钟后弃上清，得到初始微球用去离子水反复清洗三次，之后在-20℃冷冻干燥24小时，即得到纯净的粒径为10μm～100μm的生长因子缓释微球，于-20℃保存备用。
实施例2
与实施例1不同的是，将5μg的rhTGF-β3溶于200μL去离子水中，制备浓度为25μg/mL的rhTGF-β3水溶液，作为内水相w 1。
实施例3
与实施例1不同的是，将10μg的rhTGF-β3溶于200μL去离子水中，制备浓度为50μg/mL的rhTGF-β3水溶液，作为内水相w 1。
测试部分
(1)生长因子缓释微球的形态和药物分布观察
取适量实施例1-3中生长因子缓释微球置于硅片上，并经表面喷金处理，采用日本日立公司的Hitachi S-4800型扫描电子显微镜观察微球的形态特征，结果分别如图1-3所示。
图1结果表明，实施例1的生长因子缓释微球呈圆球状，形态良好，直径大小为5μm～30μm，表面较光滑，可见轻微起伏，相邻两波峰之间的间距为0.38μm～0.71μm。
图2结果表明，实施例2的生长因子缓释微球呈圆球状，形态良好，直径大小为5μm～50μm，表面较光滑，可见轻微起伏，相邻两波峰之间的间距为0.33μm～0.77μm。
图3结果表明，实施例3的生长因子缓释微球呈圆球状，形态良好， 直径大小为5μm～30μm，表面较光滑，可见轻微起伏，相邻两波峰之间的间距为0.66μm～1.98μm。
本申请实施例的生长因子缓释微球微球形貌良好，有利于细胞粘附，促进细胞的生长、迁移、增殖及再分化。
(2)生长因子缓释微球中rhTGF-β3的包封量及包封率测定
分别收集实施例1-3中微球溶液离心后的上清，使用酶联免疫吸附测定法(Enzyme-Linked ImmunoSorbent Assay，简称ELISA)测定上清液的光密度(optical density，OD)值，根据标准曲线方程分别计算上清液中rhTGF-β3的浓度及质量，再使用下列公式计算生长因子缓释微球中rhTGF-β3的包封量及包封率。共测定3次，结果取平均值。
包封率＝(微球中rhTGF-β3的质量/rhTGF-β3的总质量)×100％
包封量＝微球中rhTGF-β3的质量/微球的质量，式中包封量的单位是ng/mg，微球中rhTGF-β3的质量的单位是ng，微球的质量的单位是mg。
微球中rhTGF-β3的质量＝rhTGF-β3的总质量－上清液中rhTGF-β3的质量
结果：
实施例1中，生长因子缓释微球中rhTGF-β3的包封率平均值为：93.6％，包封量平均值为：37.5ng/mg。
实施例2中，生长因子缓释微球中rhTGF-β3的包封率平均值为：94.4％，包封量平均值为：94.4ng/mg。
实施例3中，生长因子缓释微球中rhTGF-β3的包封率平均值为：98.9％，包封量平均值为：197.8ng/mg。
可见，本申请实施例的方法中，PLGA对rhTGF-β3具有很高的包封率，并具有有效的包封量，能够提高rhTGF-β3的利用率。
(3)生长因子缓释微球的控缓释放性测定
将一定质量的生长因子缓释微球加入1mL磷酸缓冲盐溶液PBS(pH为7.4)中，之后置于转速为80rpm的室温(20℃～25℃)摇床。缓释一定时间后，10000rcf离心5分钟，取上清保存，并加入等量新鲜的PBS缓冲液(pH为7.4)继续进行缓释；在缓释周期内按此方式依次取多个上清样品。使 用ELISA试剂盒测定上清样品中rhTGF-β3的浓度并计算rhTGF-β3的累积释放量。每组测试3个样品，结果取平均值。
生长因子缓释微球的缓释结果参见表1和图4。
表1
  实施例1 实施例2 实施例3
生长因子缓释微球的加入量(mg) 24.2 8.3 4.1
第一天的缓释量(％) 0.052 0.088 0.338
第一周的缓释量(％) 0.135 0.228 0.704
实施例1至3的缓释量均是以微球的初始rhTGF-β3含量为基准。
实施例1至3的生长因子缓释微球能够实现rhTGF-β3长期有效的控缓释放，并在第一天具有最大缓释速率，且在第一周之后基本达到稳定的缓释速率。实施例1至3的生长因子缓释微球缓释期均可达到500天以上，能够实现微球中的rhTGF-β3长期逐渐缓释，从而长期发挥rhTGF-β3的细胞募集能力，满足缺损软骨组织的长期再生修复需求。
图4示出了实施例1至3的生长因子缓释微球28天体外累计释放度曲线图，其中包括缓释时间分别为1、2、4、7、14、21、28天的累计释放度。如图4所示，不同rhTGF-β3包封量的生长因子缓释微球的缓释速度不同。rhTGF-β3包封量相对较高的微球，其释放速度也相对较高。并且，不同rhTGF-β3包封量的生长因子缓释微球均没有凸释现象。
测试结果表明，本申请实施例的生长因子缓释微球rhTGF-β3释放速度缓慢，为一种良好的控释载体，达到缓慢释放rhTGF-β3、持续不断的募集干细胞和促进干细胞的软骨分化的目的，对于需要长时间生长和恢复的软骨缺陷来说提供良好的治疗效果。
(4)生长因子缓释微球Transwell迁移实验
使用康宁公司的3422型transwell小室。
实验设置6个实验组：
1)阴性组：DMEM/F12(50:50)低糖培养基，其中含有体积浓度为1％的胎牛血清FBS。DMEM/F12(50:50)低糖培养基是F12培养基(Ham’s F 12 nutrient medium动物细胞培养基)与DMEM(Dulbecco’s  Modified Eagle Medium)培养基以1:1结合。
2)阳性组：DMEM/F12(50:50)低糖培养基，其中含有1％FBS、及10ng/mL的rhTGF-β3。
3)空白组：DMEM/F12(50:50)低糖培养基，其中含有1％FBS、及10mg/mL的空白PLGA微球。
4)实验组1：DMEM/F12(50:50)低糖培养基，其中含有1％FBS、及5mg/mL实施例3的生长因子缓释微球，生长因子缓释微球的含量满足rhTGF-β3因子的含量约为1000ng/mL培养基。
5)实验组2：DMEM/F12(50:50)低糖培养基，其中含有1％FBS、及0.5mg/mL实施例3的生长因子缓释微球，生长因子缓释微球的含量满足rhTGF-β3因子的含量约为100ng/mL培养基。
6)实验组3：DMEM/F12(50:50)低糖培养基，其中含有1％FBS、及0.05mg/mL实施例3的生长因子缓释微球，生长因子缓释微球的含量满足rhTGF-β3因子的含量约为10ng/mL培养基。
具体操作步骤按说明书进行。每组样品重复3次，结果取平均值。结果如图5和6所示。
图5和图6的结果表明，本申请实施例的生长因子缓释微球具有显著的促进家兔滑膜干细胞迁移的功能，更有效地发挥了rhTGF-β3的强大细胞募集能力，能够提高缺损软骨组织的再生修复效果。
实施例4
取自兔软骨的dECM源先采用冻融法处理，之后使用质量浓度为3％的过氧化氢溶液进行处理，得到脱细胞软骨细胞外基质；将脱细胞软骨细胞外基质、实施例1的生长因子缓释微球及LAP光交联剂溶于去离子水中，制成脱细胞软骨细胞外基质和生长因子缓释微球的浓度均为0.03g/mL的第一水溶液，第一水溶液中交联剂的浓度为100μg/mL；将GelMA水凝胶溶于去离子水中，制成GelMA水凝胶的浓度为0.2g/mL的第二水溶液，其中GelMA水凝胶的甲基化程度约60％；将第一水溶液和第二水溶液按照体积比为1:1混合，得到具有打印性能的生物墨水。
采用上普公司的ALPHA-IPT1型生物3D打印机，将生物墨水加入生物打印机的与第一打印头相连的料筒中，设置保温温度为16℃；将聚己内酯颗粒加入生物打印机的与第二打印头相连的料筒中，设置保温温度为100℃；将生物打印机成型室的温度调整为5℃；按照图8和图9的结构，第一打印头与第二打印头按预设打印路径并行进行打印，打印产品经紫外光交联处理，得到组织工程软骨复合支架。
实施例5
与实施例4不同的是，生长因子缓释微球为实施例2的生长因子缓释微球。
实施例6
与实施例4不同的是，生长因子缓释微球为实施例3的生长因子缓释微球。
测试部分
(1)组织工程软骨复合支架的形态观察
实施例4中组织工程软骨复合支架的大体观如图7所示，镜下观如图10所示。图7结果表明：组织工程软骨复合支架为20mm×20mm×2mm的长方体框架结构，PCL纤维呈白色，水凝胶纤维呈透明状，孔隙中有小气泡。图10结果表明：组织工程软骨复合支架的PCL纤维的宽度约为700μm，纤维组之间的平均孔隙为500μm，水凝胶纤维的宽度约500μm。
(2)组织工程软骨复合支架的力学测定
使用美国安捷伦U9820A Nano Indenter G200型纳米力学测试系统测定
实施例4-6的组织工程软骨复合支架的压缩弹性模量。
实施例4-6的测试结果示于下面的表2。
表2
  实施例4 实施例5 实施例6
压缩弹性模量/MPa 400 400 500
结果表明：本申请实施例的组织工程软骨复合支架的压缩弹性模量可以达到400MPa～500MPa，比正常软骨的压缩弹性模量要高，能够提供较好的力学支撑。
(3)组织工程软骨复合支架细胞相容性共聚焦显微镜观察
将实施例4制备的无菌组织工程软骨复合支架与新西兰家兔第二代脂肪间充质干细胞共培养7天后，用活/死细胞染色试剂盒染色，用荧光共聚焦显微镜拍照，评估组织工程软骨复合支架的生物相容性。结果如图11所示。
在图11中，以封闭曲线标出的细胞为死细胞，剩余的是活细胞，细胞存活率可达95％左右。图11结果表明，细胞在组织工程软骨复合支架生长状态良好，细胞存活率高，说明了本申请实施例的组织工程软骨复合支架具有良好的生物相容性，有利于细胞生存、增殖及再分化。
(4)组织工程软骨复合支架细胞相容性扫描电子显微镜观察
将实施例4制备的无菌组织工程软骨复合支架与小鼠RAW细胞共培养3天后，用2.5％戊二醛固定等预处理后，在扫描电子显微镜下观察，评估组织工程软骨复合支架的生物相容性。结果如图12所示。
图12结果表明：小鼠RAW细胞在组织工程软骨复合支架生长状态良好，细胞形态多样，分泌大量细胞外基质，细胞之间相互接触，说明了本申请实施例的组织工程软骨复合支架具有良好的生物相容性，有利于细胞生存、增殖及再分化。
以上结果表明，本申请实施例提供的组织工程软骨复合支架，具有多孔结构、且生物相容性好、力学强度高，适用于软骨缺损的移植，能够很好地促进无血管区的缺损软骨组织的再生修复，提高再生修复效果。
以上所述，仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到各种等效的修改或替换，这些修改或替换都应涵盖在本申请的保护范围之内。因此，本申请的保护范围应以权利要求的保护范围为准。

Claims (14)

  1. 一种生长因子缓释微球，所述微球包括聚乳酸-羟基乙酸共聚物基质材料以及包封于所述基质材料中的重组人转化生长因子β3；
    所述微球中重组人转化生长因子β3的包封量为5ng/mg～200ng/mg；
    所述微球的缓释期为28天以上。
  2. 根据权利要求1所述的生长因子缓释微球，其中，所述微球在第一天的缓释量为0.05％～0.5％，所述微球在第一天具有最大缓释速率；
    进一步地，所述微球在第一周的缓释量为0.1％～1％。
  3. 根据权利要求1所述的生长因子缓释微球，其中，所述聚乳酸-羟基乙酸共聚物的分子量为1×10 5Da～1.1×10 5Da，优选为1.02×10 5Da；和/或，
    所述聚乳酸-羟基乙酸共聚物中乳酸与羟基乙酸的摩尔比为1:1～5:1，优选为3:1。
  4. 根据权利要求1所述的生长因子缓释微球，其中，所述微球的粒径为5μm～100μm，优选为5μm～50μm；和/或，
    所述微球的表面具有轻微起伏，相邻两波峰之间的间距为0.1μm～2.5μm，优选为0.3μm～2μm。
  5. 一种生长因子缓释微球的制备方法，包括以下步骤：
    提供浓度为1μg/mL～100μg/mL的重组人转化生长因子β3水溶液；
    提供浓度为20mg/mL～25mg/mL的聚乳酸-羟基乙酸共聚物油相溶液；
    提供浓度为5mg/mL～10mg/mL的聚乙烯醇水溶液；
    将所述重组人转化生长因子β3水溶液与所述聚乳酸-羟基乙酸共聚物油相溶液在冰浴中超声混合，得到初级乳液；
    将所述初级乳液与所述聚乙烯醇水溶液在20℃～25℃下搅拌混合，得到微球溶液；
    将所述微球溶液分离处理，得到初始微球经洗涤、冷冻干燥后，得到所述生长因子缓释微球，所述微球中重组人转化生长因子β3的包封量为5ng/mg～200ng/mg，所述微球的缓释期为28天以上。
  6. 根据权利要求5所述的制备方法，其中，所述方法还满足如下(1)～(5)中的一种或多种：
    (1)所述聚乙烯醇水溶液中聚乙烯醇的分子量为6×10 4Da～7×10 4Da，优选为6.7×10 4Da；
    (2)所述重组人转化生长因子β3水溶液与所述聚乳酸-羟基乙酸共聚物油相溶液混合的体积比为1:5～1:10，所述初级乳液与所述聚乙烯醇水溶液混合的体积比为1:5～1:25；
    (3)所述超声混合的超声功率为70W～75W，超声时间为10s～30s；
    (4)所述搅拌的速率为500rpm～1000rpm，时间为4h～12h；
    (5)所述分离处理包括将所述微球溶液在1500rpm～2000rpm下离心分离3min～5min。
  7. 一种组织工程软骨复合支架，包括：
    组织工程支架，包括沿自身高度方向依次层叠的多个支架层，相邻两个所述支架层中的一者包括多个沿第一方向相互间隔设置的纤维组，另一者包括多个沿第二方向相互间隔设置的纤维组，每个所述纤维组包括可降解高分子纤维及设置于所述可降解高分子纤维自身宽度方向的一侧或两侧的水凝胶纤维，所述水凝胶纤维沿所述可降解高分子纤维的长度方向延伸，所述第一方向与所述第二方向相交；
    基质材料，复合于所述组织工程支架的所述水凝胶纤维中；
    权利要求1至4任一项所述的生长因子缓释微球，复合于所述组织工程支架的所述水凝胶纤维中。
  8. 根据权利要求7所述的组织工程软骨复合支架，其中，所述组织工程软骨复合支架还满足如下(1)～(3)中的一种或多种：
    (1)所述支架层中，相邻所述纤维组之间的间隔为300μm～500μm，相邻所述纤维组中所述可降解高分子纤维之间的间隔为750μm～1500μm；
    (2)所述支架层的高度为100μm～500μm；
    (3)所述可降解高分子纤维的宽度为200μm～800μm，所述水凝胶纤维的宽度为200μm～800μm。
  9. 根据权利要求7所述的组织工程软骨复合支架，其中，所述组织工 程软骨复合支架还满足如下(a)～(c)中的一种或多种：
    (a)所述可降解高分子纤维为分子量为2×10 4Da～5×10 5Da的聚己内酯纤维；
    (b)所述水凝胶纤维为甲基化程度为40％～80％的甲基丙烯酸化水凝胶纤维；
    (c)所述基质材料为脱细胞软骨细胞外基质、I型胶原、II型胶原、细菌纤维素、蚕丝蛋白及糖胺多糖中的一种或多种。
  10. 根据权利要求7至9任一项所述的组织工程软骨复合支架，其中，所述组织工程软骨复合支架的压缩弹性模量为100MPa～600MPa，进一步地为200MPa～500MPa。
  11. 权利要求7至10任一项所述组织工程软骨复合支架的制备方法，包括以下步骤：
    提供包含基质材料和所述生长因子缓释微球的第一水溶液；
    提供包含水凝胶的第二水溶液；
    将所述第一水溶液和所述第二水溶液混合，得到生物墨水；
    以所述生物墨水作为所述水凝胶纤维的原料、以可降解高分子材料作为所述可降解高分子纤维的原料，打印得到所述组织工程软骨复合支架。
  12. 根据权利要求11所述的制备方法，其中，所述第一水溶液中所述基质材料的浓度为0.01g/mL～0.05g/mL，所述第一水溶液中所述生长因子缓释微球的浓度为0.01g/mL～0.05g/mL；
    所述第二水溶液中所述水凝胶的浓度为0.1g/mL～0.2g/mL；
    所述第一水溶液与所述第二水溶液混合的体积比为1:1～1:2。
  13. 根据权利要求11所述的制备方法，其中，所述以所述生物墨水作为所述水凝胶纤维的原料、以可降解高分子材料作为所述可降解高分子纤维的原料，打印得到所述组织工程软骨复合支架的步骤包括：
    将生物墨水加入生物打印机的与第一打印头相连的料筒中，设置保温温度为12℃～16℃；
    将聚己内酯加入生物打印机的与第二打印头相连的料筒中，设置保温温度为80℃～100℃；
    将生物打印机成型室的温度调整为3℃～5℃；
    所述第一打印头与所述第二打印头并行进行打印，打印产品经交联处理得到所述组织工程软骨复合支架。
  14. 根据权利要求11所述的制备方法，其中，所述第一水溶液中还含有交联剂，所述第一水溶液中所述交联剂的浓度为100μg/mL～200μg/mL。
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