CN101496908B - Pearl powder artificial bone supporting material with multi-stage micro-nano structure and technique for producing the same - Google Patents
Pearl powder artificial bone supporting material with multi-stage micro-nano structure and technique for producing the same Download PDFInfo
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Abstract
The invention relates to a novel biological scaffold material for bone repair and a preparation method thereof. PLGA and PLA with a mass ratio of 1: 1-10: 1 are dissolved into chloroform, dimethyl sulfoxide, 1, 4-dioxane or a mixed liquid of the 1, 4-dioxane and ultrapure water, and then pearl powder which is subjected to partial or complete deproteinization treatment is added into the obtained product according to the proportion of the PLGA/a PLA mixture to the pearl powder (mass ratio) is 1: 1-10: 1 to obtain forming slurry. A three-dimensional scaffold with high porosity and connectivity rate is designed through a 3D software, and then a low-temperature rapid forming system is utilized to ensure that the three-dimensional scaffold is formed to obtain a scaffold with a microporous structure. An artificial bone biological scaffold material prepared by the method has a three-dimensional scaffold structure that a macroscopic structure has aperture channels with diameters of between 100and 500mu m and a microstructure has micropores with diameters of between 10 and 20mu m, wherein micron pearl powder is dispersedly distributed on walls of the micropores. The porosity is between 60 and 90 percent, and a macroporous structure is perforated in three directions of X axis, Y axis and Z axis, and has 100 percent of connectivity.
Description
Technical field
The invention belongs to the bio-medical material technical field, specifically, relate to a kind of new biologic bracket material that is used for the bone reparation and preparation method thereof.
Background of invention
At present in the world, do not have rejection, be used for the artificial bone tissue of bone defect repair, have urgent and great demand having good physiological function.Make the field at artificial bone, foreign study mechanism and enterprise have dropped into ample resources in recent years, and have obtained a series of achievements in research, and some artificial bones that are used for defect repair begin to get into clinical practice.Meanwhile, the key problem of bone holder material such as biocompatibility, osteoinductive, porosity and mechanical strength etc. still exist a lot of problems to need to solve.
Margarita powder is a kind of ideal natural osteogenic materials, has biocompatibility, characteristics that inductivity is good, has broad application prospects.The physicochemical property of nacreous layer and biological characteristics and osseous tissue are very approaching, and research shows between nacreous layer and the bone to have sibship on the hereditism, makes nacreous layer have excellent biological compatibility (Westbroek P, et al; 1998,392 (6679): 861-2).And the nacreous layer implant in vivo can be dissolved, absorb the biodegradation character that tool is good.In vivo nacreous layer is injected vertebral body bone lacks model; Can be observed nacreous layer degrades gradually; The bone defect forms new ripe bone trabecula, and the bone trabecula surface is covered by linearly aligned osteoblast osteoid, and nacreous layer is combined closely with new bone; Show that nacreous layer can break up and new bone formation (Lamghari M, et al by stimulating osteoblast in vivo; Biomaterials.2001,22 (6): 555-62).Research to mechanism shows, discharges calcium ion during the nacreous layer degraded, and the local Ca ion concentration is increased, and forms the prerequisite of skeletonization mineralising.Miyamoto H etc. (Proc.Natl.Acad.Sci.U S A, 1996,93 (18): but 9657-60) think nacreous layer stromatin N66 carbonic anhydrase spline structure territory catalysis HCO3-form, participate in the formation of calcium carbonate crystal.And (FEBS Lett, 1999,462 (1-2): 225-9) find that stromatin N16, N66 and N4 duplicate block can form the structure of similar glycine ring appearance, have combination Ca such as Samata T
2+Ability, the regulation and control crystallization forms.Nacreous layer substrate also can produce some chemical signals, has the osteoblastic ability of activation (Almeida MJ, et al; J.Biomed.Mater.Res.2001,57 (2): 306-12).Warps such as Lopez are tested for many years and research is thought and contained the signaling molecule that is similar to BMPs (bone morphogenetic protein) in the Concha Margaritifera organic principle; After getting in the body, can be spread in the bone marrow, then the skeletonization stromal cell is produced a kind of chemotaxis; Bone growth promoting cell in the activated bone marrow; Make its differentiation, produce osteoblast, finally induce new bone formation.Zhang En etc. (the mineral journal, 2008,2:112-116) adopt SEM and HRTEM scientific discovery pearly layer to form the lamellar crystal layer by the agglutinating aragonite of organic matter, and with the mutual parallel stacked of organic horizon.Simultaneously, on prism-shaped and the crystalline cross section of lamellar aragonite, contain organic matter, gas liquid inclusion or the hole of shapes such as a large amount of nano level circles, ellipse.
Margarita powder is as artificial bone, the key issue that solves of still needing be material plasticity and insufficient formability, difficult formation has the structure that fine porosity and UNICOM lead.And methods such as common chemical foaming, high temperature sintering are poor to the molding effect of Margarita powder; Also cause easily the active component that has bone inductive effect in the Margarita powder is lost activity; Therefore select suitable and advanced porous support manufacturing process; Comprising the selection of adjuvant and forming method, is the key issue that can the Margarita powder material effectively be applied to the bone reparative regeneration.
Summary of the invention
An object of the present invention is to provide a kind of new compositions that is used to prepare the artificial bone biologic bracket material.
Another object of the present invention provides a kind of artificial bone biologic bracket material, and this material has high porosity and high connection rate, has mechanical strength preferably simultaneously.
Another object of the present invention provides a kind of method for preparing above-mentioned artificial bone biologic bracket material.
Margarita powder is as artificial bone, and the key issue that solves of still needing is material plasticity and insufficient formability, and difficult formation has the structure of fine porosity and connection rate.And polylactic acid (Polylactic acid; PLA) be the degradable high polymer material that is polymerized by lactide (LA) under certain condition; (polylactide-co-glycolide PLGA) then is the degradable high polymer material that is polymerized by lactide (LA) and Acetic acid, hydroxy-, bimol. cyclic ester (GA) under certain condition to polylactic acid/ethanol copolymer.These two kinds of materials have the plasticity height, and degraded is fast, are prone to the characteristics that are shaped; Have excellent biological compatibility and bone conduction performance; Can carry out biodegradation completely, its catabolite can get into the metabolic pathway of human body, finally is converted into carbon dioxide and water; The FDA approval of these two kinds of materials is widely used in the adjuvant of slow releasing preparation such as injection microcapsule, microsphere and implants, can be used as the porous support of organizational project cell culture simultaneously.
The inventor finds through a large amount of experiments, is that molding adjuvant and Margarita layer powder are compound with biocompatible macromolecular material PLA and PLGA, can remedy the deficiency that it is difficult for shaping, simultaneously porosity, aperture and the mechanical strength of scalable molding support.
Therefore, the inventor has prepared a kind of new compositions, and said composition comprises PLGA, PLA and Margarita powder, and PLGA in the said compositions: PLA (mass ratio) is 1: 1-10: 1, preferred 3: 1-5: 1; The PLGA/PLA mixture: Margarita powder (mass ratio) is 1: 1-10: 1, preferred 2: 1-4: 1, most preferably be 7: 3; Described Margarita powder granularity is below 2000 orders, and preferred diameter is the 1-10 micron, more preferably the 3-6 micron.
In the above-mentioned composition, PLGA: PLA (mass ratio) is 1: 1-10: 1, preferred 3: 1-5: 1.The ratio of LA/GA is 90/10,80/20,75/25,60/40 or 50/50 among the described PLGA, and preferred LA/GA is 50/50.In said composition, the adding of PLA is in order to regulate the mechanical strength of the artificial bone biologic bracket material of processing at last, to increase stability and mechanical property requirements that its ratio can be improved supporting structure.
PLGA, the PLA of aforementioned proportion are dissolved in organic solvent; Organic solvent can select 1,4-dioxane or 1, the mixing material of 4-dioxane and ultra-pure water; Preferred 1; The mixing material of 4-dioxane and ultra-pure water, ultra-pure water accounts for the 1%-20% of cumulative volume in the mixing material, is preferably 5%-10%.After PLGA, PLA be dissolved in organic solvent, then according to the PLGA/PLA mixture: Margarita powder (mass ratio) was with 1: 1-10: 1, preferred 2: 1-4: 1, and the ratio that most preferably is 7: 3 adds the shaping slurry that Margarita powder becomes preparation artificial bone biologic bracket material.Wherein, described Margarita powder granularity is below 2000 orders, and preferred diameter is the 1-10 micron, more preferably the 3-6 micron.For improving the biocompatibility of Margarita powder, reduce immunogenicity, the Margarita powder of above-mentioned Margarita powder preferred part Deproteinization Margarita powder or complete Deproteinization.The biocompatibility of handling the back Margarita powder through special part deproteinization can further improve, and keeps the biotic factor with bone-inducting active simultaneously.Complete Deproteinated Margarita powder then keeps other regular sheet lamellar spacing of 100nm level and circular opening structure, and this structure helps the generation of nutrient substance diffusion, osteoblastic proliferation and bone.
Above-mentioned shaping slurry can be used for preparing the artificial bone biologic bracket material; Uniform polyester liquid generation solid-liquid is separated/liquid-liquid phase separation in the slurry in low temperature environment; Generate solvent crystallization and the crystallization mutually of rich polyester, the isolating biphase solid that all is frozen under lower temperature then.Organic solvent volatilizees rapidly in the vacuum lyophilization environment, the final compositions that contains PLGA, PLA and Margarita powder that forms, and PLGA in the said compositions: PLA (mass ratio) is 1: 1-10: 1, preferred 3: 1-5: 1; The PLGA/PLA mixture: Margarita powder (mass ratio) is 1: 1-10: 1, preferred 2: 1-4: 1, most preferably be 7: 3; Described Margarita powder granularity is below 2000 orders, and preferred diameter is the 1-10 micron, more preferably the 3-6 micron.
In bone is repaired, clinical needs are the most urgent be have larger volume, specific shape arranged, baroque bone holder material.Good porosity is the quick vascularization of tissue engineered bone, and by from the alternate basis of body bone.For big section bone holder material manufacturing, methods such as common burning, foaming all can not meet the demands on porosity and mechanical property, and rapid shaping (RPM) manufacturing technology is the selection of at present tool prospect.Because rapid shaping technique to the controllability of supporting structure, makes to the possibility that is designed to of supporting structure, and structure not only influences porosity, connection rate, the vascularization of artificial bone, has also determined the mechanical property of material.The researcher of U.S. MIT and Princeton University, the successful use rapid forming equipment has produced artificial aggregate in recent years, and at present product has been accomplished zoopery, gets into to examine (Sherwood JK, et al before clinical; Biomaterials, 2002,23 (24): 4739-51).(J.Biomed.Mater.Res.B.Appl.Biomater such as Germany Seitz H; 2005,74 (2): 78-28) use the 3D printing technique, successfully made inside and had the bone material of complex passages and hole, material presents excellent mechanical intensity, begins to have got into industrialization development at present.
Utilize aforesaid shaping slurry, adopt method can make up artificial bone biologic bracket material with high porosity and connection rate with the low temperature rapid shaping.The above-mentioned method that is used for the Biocomposite material support of bone reparation of the preparation that the present invention proposes may further comprise the steps:
(1), preparation part deproteinization Margarita powder or deproteinization Margarita powder fully;
(2), preparation contains PLGA, the slurry of PLA and Margarita powder;
(3), the three-dimensional rack that has high porosity and connection rate through the 3D software design;
(4), utilize the low temperature rapid prototyping system to make the three-dimensional rack molding.
In step (1); It is dry to get the micron order Margarita powder of granularity below 2000 orders; Use vibrosieve that it is sieved to obtain preferred diameter as 1-10 micron, the Margarita powder of 3-6 micron more preferably then, subsequently Margarita powder is carried out defat, surperficial decalcification; Part deproteinization or deproteinization processing fully, the sterilization of dry back is subsequent use.
In step (2), be 1 with mass ratio: 1-10: 1 (preferred 3: 1-5: 1) PLGA and PLA are dissolved in organic solvent, and said organic solvent is selected from chloroform, dimethyl sulfoxide, 1; 4-dioxane or 1; The mixing material of 4-dioxane and ultra-pure water, preferred 1, the mixing material of 4-dioxane and ultra-pure water; Ultra-pure water accounts for the 1%-20% of cumulative volume in the mixing material, is preferably 5%-10%.After PLGA, PLA be dissolved in organic solvent; It is subsequent use to process the shaping slurry according to the Margarita powder in certain ratio adding step (1) then; Described PLGA/PLA mixture: Margarita powder (mass ratio) is 1: 1-10: 1, preferred 2: 1-4: 1, most preferably be 7: 3;
In bone is repaired, histiocyticly grow into, the exchange of generation, nutrient and the metabolite of neovascularity and the degraded of timbering material all require support to have high porosity and connection rate.The brace aperture rate is too low, and surface area is too little, and then the connectivity between the hole is bad, and what be unfavorable for organizing grows into; Since little with the contact area of body fluid, also be unfavorable for support degraded in vivo.Therefore, under the prerequisite of the mechanical property of stability that guarantees supporting structure and requirement, support should have try one's best high porosity and connection rate.In step (3), the support model that has high porosity and connection rate with 3D software (for example Solidworks) design; According to actual needs, utilize the support model of 3D software design arbitrary shape, the cad model size that designs is saved as stl file.Open the model stl file with Aurora software, after stl file is tested and revised, adopt the macroporous structure unit that it is carried out three-dimensional and fill and handle, make up and contain regular aperture 100～500 μ m macroporous structures, three directions perforations of the XYZ axle of macroporous structure; Filling to the stl file after filling carries out in layering and the synusia is handled; After check/correction, convert to can the drive mechanism body subsystem sweep vector.
On the basis of Yin Hua company rapid prototyping system, install low temperature moulding chamber and temperature control system additional.In hierarchical information file input Cark shaping control software, generate information such as coordinate pathway automatically; The driving system of stepping motor of motor control card control X, Y, three directions of Z, control biomaterial molding shower nozzle shaping position, supercharging shower nozzle under driver element control, stable extrusion molding slurry.Indoor at cryogenic forming, the rapid cooling of slurry, in the slurry uniformly polyester liquid generation solid-liquid be separated/liquid-liquid phase separation, generate solvent crystallization and rich polyester mutually, then the isolating biphase solid that all is frozen under lower temperature.The freezing support that from the cryogenic forming chamber, takes out is directly put into freezer dryer and is removed solvent and backing material.In the vacuum environment in freezer dryer, the solvent in the freezing support distils, and obtains having the support of microcellular structure.This microcellular structure of support directly depends on the two phase structure that forms in the phase separation, and solvent crystallization forms hole mutually mutually, and rich polyester forms hole wall mutually, and the Pearl disperse is distributed in the hole wall.
Artificial bone biologic bracket material according to method for preparing; Its macrostructure is for having 100-500 μ m aperture passage; Micro structure is the three-dimensional rack structure with 10-20 μ m micropore; The micro-pore wall disperse micron order Margarita powder that distributing wherein, Margarita powder has other regular sheet lamellar spacing of 100nm level and circular opening structure.The brace aperture rate is 60%-90%, and the X of macroporous structure, Y, three directions of Z axle connect, and have 100% perforation rate; This artificial bone biologic bracket material is that osteoblast provides the three dimensions of growth and enough surfaces externally and internallies to amass; Helping cell suspension immerses and sticks; Obtain enough nutrient substance, carry out gas exchange, discharge metabolite; Make the three dimensions growth of cell, finally form osseous tissue by prefabricated form.
The present invention as the composite of matrix compound pearl powder and the supporting structure that obtains with the low temperature rapid shaping technique, has following major advantage with the PLGA/PLA material:
1. Margarita powder has excellent biological compatibility, inductivity, and the calcium ion that produces during the Margarita powder degraded can be utilized by freshman bone tissue, promotes the regeneration of osseous tissue; But differentiation of the albumen stimulating osteoblast in the nacreous layer and propagation activate the osteoblast activity, participate in the formation of calcium carbonate crystal; The biocompatibility of Margarita powder further improves after special part deproteinization is handled, and the active factors with bone-inducting active obtains keeping.Complete Deproteinated Margarita powder then keeps other regular sheet lamellar spacing of 100nm level and circular opening structure, helps the generation of nutrient substance diffusion, osteocyte propagation and bone.
2. add the bonding agent that PLGA/PLA serves as Margarita powder, make shaping of material more convenient, adopt the low temperature rapid shaping technique, kept away other violent method and caused Margarita powder to induce bone active forfeiture, the destruction of regular nanostructured in the Margarita powder.The brace aperture rate that makes up is 60%-90%.The support overall structure is accomplished designing requirement, and support has the macroporous structure and the passage of rule, and the X of macroporous structure, Y, three directions of Z axle connect, and have 100% perforation rate;
3. it is the key request of a support that enough mechanical performances are provided; If support can not be provided at the mechanical modulus of a series of hard tissues (10-1500MPA) or soft tissue (0.4-350MPA), the shaping of any one cambium also may excessive deformation and is failed so.The supporting structure that the present invention makes; Measured value be respectively modulus of elasticity in comperssion 17.83 ± 1.73MPa-25.62 ± 2.35MPa and comprcssive strength 0.67 ± 0.07MPa-1.47 ± 0.11MPa; Composite has than the higher intensity of single PLGA/PLA and than single Margarita powder better toughness, and comprehensive mechanical property has obtained optimization.
4. the design of hierarchy; Satisfied the steric requirements of osteogenesis; The macroporous structure rule is mellow and full; Aperture 100～500 μ m help induction of vascular and the ripe bone that contains haversian system to grow into, and are covered with mellow and full microcellular structure below the 100 μ m of aperture in the hole wall of macropore and help Margarita powder and other somatomedin of cell mass growth and compound disperse distribution and realize controllable sustained-release; The pore wall thickness of micropore is 1-10 μ m, and other regular sheet lamellar spacing of Margarita powder 100nm level and circular opening structure help the generation of nutrient substance diffusion, osteoblastic proliferation and bone, can increase the bone-inducting active of support
Below in conjunction with the specific embodiment the present invention is described further, protection scope of the present invention is not limited to embodiment, every according in disclosed by the invention perhaps principle any this area of implementing be equal to replacement, all belong to protection scope of the present invention.
Description of drawings
Fig. 1 is low temperature rapid molding device structure and shower nozzle forming process figure, on the basis of Yin Hua company rapid prototyping system, installs low temperature moulding chamber and temperature control system additional.The space size of working chamber is 300mm (directions X) * 320mm (Y direction) * 700mm (Z direction), and open-top, refrigerating plant are distributed in a side of working chamber, and opposite side has openable actuating doors, and minimum cryogenic temperature is-40 ℃, and the constant temperature ability is ± 2 ℃.Wherein, (1) is Y axle slide block among Fig. 1, and (2) are X axle slide block, and (3) are storage tank; (4) be Z axle slide block, (5) are the shower nozzle fixed mount, and (6) are feeding tube, and (7) are heat insulation mantle; (8) be the low temperature moulding chamber, (9) are shower nozzle, and (10) are molded support, and (11) are workbench.
Fig. 2 is three-dimensional rack multilevel hierarchy figure, and wherein 2-A is an overall structure; 2-B is a cross section macropore array structure; 2-C is macroporous structure (ultramicroscope); 2-D is microcellular structure (ultramicroscope).
Fig. 3 is that PLGA/PLA, Margarita powder composite material bracket and NZw mesenchymal stem cells MSCs are cultivated the electron microscope picture after 10 days altogether, and demonstration MSC cell is bred in support in a large number.
Accompanying drawing 4 was cultivated 10 days for PLGA/PLA, Margarita powder composite material bracket and NZw mesenchymal stem cells MSCs altogether, and the evaluation figure of anti-I type collagen antibody immuning tissue, picture background are green.
Accompanying drawing 5 was cultivated 21 days for PLGA/PLA, Margarita powder composite material bracket and NZw mesenchymal stem cells MSCs altogether, and the calcium tuberosity figure of generation is induced in day alizarin red dyeing, and background is a black among the figure, and speckle is red calcium tuberosity.
The specific embodiment
Get Margarita powder (Zhejiang Shan Xiahu Margarita group), it carried out the screening of-2000 orders and+3000 purposes to obtain the Margarita powder of diameter at 1 μ m-10 μ m with vibrosieve, subsequently to Margarita powder with 1: 1 chloroform/methanol defat 12h; Tri-distilled water rinsing behind 37 ℃ of PBS+NaOH+NaN3 mixed liquid dipping 12h; 37 ℃ of 200g/L H
2O
2Soak 6h with the part deproteinization; Tri-distilled water rinsing 6 times repeatedly, it is dry to put into 37 ℃ of thermostatic drying chambers; Pack, cobalt 60 sterilizations are subsequent use.
PLGA/PLA (3: 1) is dissolved in 1 according to the concentration of 15% (w/v), 4-dioxane (chemical reagent purchase and supply 5-linked chemical plant, Shanghai, analytical pure) organic solvent, and 40 ℃ of stirring and dissolving, the cloud point of the liquid-liquid phase separation system of formation is 28 ℃; According to PLGA/PLA: the ratio of Margarita powder=70/30 (w/w) adds preparation shaping slurry behind the Pearl; 30 ℃ of room temperature are preserved subsequent use.
Make up the three dimensional structure that designs, cad model size: 2 * 2 * 2cm with 3D software Solid works
3Open 3D support model stl file with Aurora software, after stl file is tested and revised, adopt the macroporous structure unit that it is carried out three-dimensional and fill processing; Structure contains regular aperture 200 μ m macroporous structures, and three directions of the XYZ axle of macroporous structure connect; Filling to the stl file after filling carries out in layering and the synusia is handled, and generation synusia file (SLI, CLI) and grid file (NET) mesh width: d=1mm; After check/correction, convert to can the drive mechanism body subsystem the molding of sweep vector driving arrangement (Yin Hua company rapid prototyping system, Fig. 1).The low temperature moulding room temperature is-30 ℃; Shower nozzle rotating speed controlled frequency is 200Hz; Scanning speed is 6mm/s.
Indoor at cryogenic forming, the rapid cooling of slurry, in the slurry uniformly polyester liquid generation solid-liquid be separated/liquid-liquid phase separation, generate solvent crystallization and rich polyester mutually, then the isolating biphase solid that all is frozen under lower temperature.The freezing support that from the cryogenic forming chamber, takes out is directly put into freezer dryer and is removed solvent and backing material (40 ℃).In the vacuum environment in freezer dryer, the solvent in the freezing support distils, and obtains having support (Fig. 2-A, Fig. 2-B) of microcellular structure.This microcellular structure of support directly depends on the two phase structure that forms in the phase separation, and solvent crystallization forms hole mutually mutually, and rich polyester forms hole wall mutually, and the Pearl disperse is distributed in the hole wall.The macrostructure of support has 100-500 μ m aperture passage, and (Fig. 2-C), micro structure is for having 10-20 μ m micropore (the three-dimensional rack structure of Fig. 2-D), wherein the micro-pore wall disperse micron order Margarita powder that distributing.Porosity is 79%, and the X of macroporous structure, Y, three directions of Z axle connect, and have 100% perforation rate; Support modulus of elasticity in comperssion 18.71 ± 1.61MPa and comprcssive strength 0.92 ± 0.06MPa.
Get Margarita powder (Zhejiang Shan Xiahu Margarita group), vibrosieve is carried out 2000 purposes screenings to obtain the Margarita powder of diameter at 3-6 μ m to it, subsequently to Margarita powder with 1: 1 chloroform/methanol defat 12h; Tri-distilled water rinsing behind 37 ℃ of PBS+NaOH+NaN3 mixed liquid dipping 12h; 37 ℃ of 200g/L H
2O
2Soak 24h with complete deproteinization; Tri-distilled water rinsing 6 times repeatedly, it is dry to put into 37 ℃ of thermostatic drying chambers; Pack, the Co60 illumination-based disinfection is subsequent use.
3D software Solid works makes up the three dimensional structure that designs, cad model size: 1.5 * 1.5 * 5cm
3Open 3D support model stl file with Aurora software, after stl file is tested and revised, adopt the macroporous structure unit that it is carried out three-dimensional and fill processing; Structure contains regular aperture 200 μ m macroporous structures, and three directions of the XYZ axle of macroporous structure connect; Filling to the stl file after filling carries out in layering and the synusia is handled, and generation synusia file (SLI, CLI) and grid file (NET) mesh width: d=0.5mm; After check/correction, convert to can the drive mechanism body subsystem the molding of sweep vector driving arrangement.Forming room's temperature is-30 ℃; Shower nozzle rotating speed controlled frequency is 250Hz; Scanning speed is 5mm/s.
Indoor at cryogenic forming, the rapid cooling generation solid-liquid of slurry is separated/liquid-liquid phase separation, generates solvent crystallization and rich polyester mutually, then the isolating biphase solid that all is frozen under lower temperature.The freezing support that from the cryogenic forming chamber, takes out is put into liquid nitrogen earlier and is preserved 10min, puts into freezer dryer then and removes solvent and backing material (40 ℃).In the vacuum environment in freezer dryer, the solvent in the freezing support distils, and obtains having the support of microcellular structure.The macrostructure of support has 100 μ m aperture passages, and micro structure is the three-dimensional rack structure with 10-20 μ m micropore, wherein the micro-pore wall disperse micron order Margarita powder that distributing.Porosity is 85%, and the X of macroporous structure, Y, three directions of Z axle connect, and have 100% perforation rate; Support modulus of elasticity in comperssion 19.88 ± 1.87MPa and comprcssive strength 0.98 ± 0.08MPa.
Embodiment 3
Get the composite material bracket and the NZw mesenchymal stem cells MSCs (MSCs) of embodiment 1 preparation, be incubated at three-dimensional cell/tissue culture system (RCCS altogether
TM, NASA), to cultivate after 10 days, 4 ℃ of glutaraldehydes are fixing, with behind 4 ℃ of the acetic acid fixedly behind the 1h, acetic acid uranium-lead citrate double staining, sem observation structure (Fig. 3).Showed cell is grown, breeds, moves good in the three-dimensional rack structure, and cell stretches according to certain direction in three dimensions, and in hole, forms RF, and stays the passage that nutrient substance passes through.
Get the composite material bracket and the NZw mesenchymal stem cells MSCs (MSCs) of embodiment 1 preparation, be incubated at three-dimensional cell/tissue culture system (RCCS altogether
TMNASA), cultivate after 10 days, take out support; Fixing 30 minutes of 10% neutral formalin solution; Anti-I type collagen antibody with the FITC labelling carries out immuning tissue's evaluation, fluorescence microscope (Fig. 4), and visible cell and structure more than 90% expressed green fluorescence.Type i collagen accounts for 90% of osseous tissue content of organics, and the MSC cell is divided into osteoblast, the important component of synthetic skeleton in a large number in the research proof support.
Get the composite material bracket and the NZw mesenchymal stem cells MSCs (MSCs) of embodiment 1 preparation, be incubated at three-dimensional cell/tissue culture system (RCCS altogether
TMNASA), cultivate after 21 days, take out support, fixing 30 minutes of 4% paraformaldehyde, the calcium tuberosity of generation is induced in day alizarin red dyeing, and a large amount of red calcium tuberositys appear in the position of the inherent cell aggregation of visible support, and support presents significant bone-inducting active (Fig. 5).
Claims (27)
1. compositions that contains PLGA, PLA and Margarita powder, it is characterized in that: PLGA in the said compositions: the PLA mass ratio is 1: 1-10: 1; The PLGA/PLA mixture: the Margarita powder mass ratio is 1: 1-10: 1, and the ratio of the LA/GA of PLGA is 90/10,80/20,75/25,60/40 or 50/50 in the said compositions.
2. compositions according to claim 1 is characterized in that: PLGA in the said compositions: the PLA mass ratio is 3: 1-5: 1.
3. compositions according to claim 1 is characterized in that: PLGA/PLA mixture in the said compositions: the Margarita powder mass ratio is 2: 1-4: 1.
4. compositions according to claim 3 is characterized in that: PLGA/PLA mixture in the said compositions: the Margarita powder mass ratio is 7: 3.
5. compositions according to claim 1 is characterized in that: described Margarita powder granularity is below 2000 orders.
6. compositions according to claim 1 is characterized in that: described Margarita powder diameter is the 1-10 micron.
7. compositions according to claim 6 is characterized in that: described Margarita powder diameter is the 3-6 micron.
8. compositions according to claim 1 is characterized in that: Margarita powder is part deproteinization or complete Deproteinated Margarita powder in the said compositions.
9. compositions according to claim 1 is characterized in that: the ratio of the LA/GA of PLGA is 50/50 in the said compositions.
10. artificial bone biologic bracket material; It is characterized in that: described timbering material is that macrostructure has 100-500 μ m aperture passage; Micro structure is the three-dimensional rack structure with 10-20 μ m micropore; Said timbering material gets PLGA in the said compositions by the preparation of compositions that contains PLGA, PLA and Margarita powder: the PLA mass ratio is 1: 1-10: 1; The PLGA/PLA mixture: the Margarita powder mass ratio is 1: 1-10: 1.
11. artificial bone biologic bracket material according to claim 10 is characterized in that: the micro-pore wall disperse micron order Margarita powder that distributing in the described timbering material.
12. artificial bone biologic bracket material according to claim 10 is characterized in that: the porosity of described timbering material is 60%-90%.
13. artificial bone biologic bracket material according to claim 10 is characterized in that: the X of the macroporous structure of described timbering material, Y, three directions of Z axle connect, and have 100% perforation rate.
14. artificial bone biologic bracket material according to claim 10 is characterized in that: the modulus of elasticity in comperssion of described timbering material is 17.83 ± 1.73MPa-25.62 ± 2.35MPa.
15. artificial bone biologic bracket material according to claim 10 is characterized in that: the comprcssive strength 0.67 ± 0.07MPa-1.47 ± 0.11MPa of described timbering material.
16. a method for preparing artificial bone biologic bracket material as claimed in claim 10 comprises the steps:
(1) antigen Margarita powder or complete Deproteinization Margarita powder are removed in preparation;
(2) preparation contains the slurry of PLGA, PLA and Margarita powder;
(3) has the three-dimensional rack of high porosity and connection rate through the 3D software design;
(4) utilize the low temperature rapid prototyping system to make the three-dimensional rack molding.
17. method according to claim 16; It is characterized in that: the process of step (1) is to get the micron order Margarita powder drying of granularity below 2000 orders; With vibrosieve it is sieved to obtain the Margarita powder of diameter at 1 μ m-10 μ m then; Subsequently Margarita powder is carried out defat, part deproteinization or deproteinization processing fully, the sterilization of dry back is subsequent use.
18. method according to claim 16; It is characterized in that: the process of step (2) is for being 1 with mass ratio: 1-10: 1 PLGA and PLA are dissolved in the organic solvent; Described organic solvent is selected from chloroform, dimethyl sulfoxide, 1; 4-dioxane or 1, the mixing material of 4-dioxane and ultra-pure water, then according to the PLGA/PLA mixture: the Margarita powder mass ratio is 1: 1-6: the Margarita powder that 1 ratio adds in the step (1) is processed the shaping slurry.
19. method according to claim 18 is characterized in that: PLGA in the process of step (2): the PLA mass ratio is 3: 1-5: 1.
20. method according to claim 18 is characterized in that: PLGA/PLA mixture in the process of step (2): the Margarita powder mass ratio is 2: 1-4: 1.
21. method according to claim 20 is characterized in that: PLGA/PLA mixture in the process of step (2): the Margarita powder mass ratio is 7: 3.
22. method according to claim 18 is characterized in that: organic solvent is 1 in the process of step (2), the mixing material of 4-dioxane and ultra-pure water, and ultra-pure water accounts for the 1%-20% of cumulative volume in the mixing material.
23. method according to claim 22 is characterized in that: organic solvent is 1 in the process of step (2), the mixing material of 4-dioxane and ultra-pure water, and ultra-pure water accounts for the 5%-10% of cumulative volume in the mixing material.
24. method according to claim 16 is characterized in that: used 3D software is Solidworks in the step (3), can design the support model of arbitrary shape according to actual needs.
25. method according to claim 24 is characterized in that: the support model of design contains the regular mellow and full macroporous structure of 100～500 μ m in the step (3), and three directions of the XYZ axle of macroporous structure connect.
26. method according to claim 16 is characterized in that: the low temperature rapid prototyping system described in the step (4) installs low temperature moulding chamber and temperature control system additional on the basis of Yin Hua company rapid prototyping system.
27. method according to claim 26 is characterized in that: the process of step (4) generates information such as coordinate pathway automatically in the control software that hierarchical information file input Cark is shaped; The driving system of stepping motor of motor control card control X, Y, three directions of Z, control biomaterial molding shower nozzle shaping position, supercharging shower nozzle under driver element control, stable extrusion molding slurry; Indoor at cryogenic forming, the rapid cooling of slurry, in the slurry uniformly polyester liquid generation solid-liquid be separated/liquid-liquid phase separation, generate solvent crystallization and rich polyester mutually, then the isolating biphase solid that all is frozen under lower temperature; The freezing support that from the cryogenic forming chamber, takes out is directly put into freezer dryer and is removed solvent and backing material; In the vacuum environment in freezer dryer, the solvent in the freezing support distils, and obtains having the support of microcellular structure.
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| CN101856515B (en) * | 2010-06-04 | 2013-03-13 | 西南大学 | Method for preparing artificial bone from chitosan and shell powder serving as raw materials |
| CN102357262A (en) * | 2011-10-09 | 2012-02-22 | 清华大学 | Porous composite scaffold of PLLA (polylactic acid)/pearl powder and its preparation method |
| CN102416198A (en) * | 2011-12-02 | 2012-04-18 | 清华大学 | Preparation method for pearl active ingredient extract/hydroxyapatite bone repair material |
| CN102512266B (en) * | 2012-01-16 | 2015-04-29 | 杭州电子科技大学 | Method for preparing spinal cord injury repair tissue engineering stent |
| CN102784014B (en) * | 2012-08-14 | 2014-11-26 | 中国科学院深圳先进技术研究院 | Porotic bone scaffold and preparation method thereof |
| CN102949753A (en) * | 2012-11-26 | 2013-03-06 | 中国人民解放军第四军医大学 | Porous composite bracket and preparation method thereof |
| CN103480045B (en) * | 2013-09-10 | 2015-09-16 | 黄晚兰 | Fully-degradable polymer material cardiovascular stent |
| FR3016293B1 (en) * | 2014-01-10 | 2019-12-20 | Mbp (Mauritius) Ltd | METHOD FOR MANUFACTURING OSTEOSYNTHESIS DEVICES, OSTEOSYNTHESIS DEVICES AND IMPLANTS OF HYBRID SEMI-SYNTHETIC MATERIAL OBTAINED BY STRUCTURAL MODIFICATION OF THE COMPONENTS OF A NATURAL MARINE BIOMATERIAL |
| CN105727368B (en) * | 2016-01-08 | 2019-06-25 | 深圳市第二人民医院 | A kind of three-dimensional composite material bracket and preparation method thereof |
| CN107041971A (en) * | 2016-09-19 | 2017-08-15 | 盐城工业职业技术学院 | A kind of fibroin based on 3 D-printing/gelatin timbering material and preparation method thereof |
| CN107185048B (en) * | 2017-06-28 | 2020-08-04 | 海口市人民医院 | Preparation method of nano pearl powder/C-HA composite stent |
| CN107261215B (en) * | 2017-06-28 | 2020-04-21 | 海口市人民医院 | Nano pearl powder/C-HA composite stent and application thereof |
| CN108904103A (en) * | 2018-05-30 | 2018-11-30 | 中国科学院深圳先进技术研究院 | A kind of method and apparatus preparing heterogeneous bone renovating material |
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