CN102319449B - Poly(lactic-co-glycolic acid)-based growth factor gradient release microsphere stent as well as preparation method and application thereof - Google Patents
Poly(lactic-co-glycolic acid)-based growth factor gradient release microsphere stent as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a poly(lactic-co-glycolic acid)-based growth factor gradient release microsphere stent. The microsphere stent is prepared by a method comprising the following steps: firstly, preparing a poly(lactic-co-glycolic acid) (PLGA) homogeneous microsphere and a transforming growth factor (TGF)-beta3 and bone morphogenetic protein (BMP)-2/PLGA microsphere; dispersing the two microspheres into ddH2O to prepare suspension; injecting the suspension into a die with a filter device at the bottom; filtering by adopting the ddH2O, and regulating the position of the ddH2O in the die; accumulating the microspheres in the die; and sintering the microspheres by adopting absolute ethanol. According to the microsphere stent prepared in the invention, the original excellent biocompatibility of the PLGA is maintained, and the mechanical performance, osteochondral induction effect and seed cell carrying capability of the microsphere stent are remarkably improved. The microsphere stent has excellent biocompatibility, can carry an enough amount of seed cells, and is used for treatment of osteochondral defects caused by tumors, trauma, severe sepsis, congenital malformation and other various diseases.
Description
Technical field
The present invention relates to biological repair composite material of a kind of bone cartilage and its preparation method and application, be specifically related to a kind of somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer and discharge microsphere support and its preparation method and application.
Background technology
Along with the development of economic and social life, the sickness rate of osteochondral defect rises year by year, and according to conservative estimation, the U.S. annual nearly 7,000,000 goes to a doctor because of orthopaedic disease, and medical treatment consumes about 2,150 hundred million dollars.China is also numerous because of the osteoarthritis that Different types of etiopathogenises is brought out every year, consume significant.The articular prosthesis of present clinical employing is replaced into one of effective Therapeutic Method.But the operating substitution joint is not only expensive, and complication danger is arranged.It is that new way and new method have been opened up in the reparation of cartilage defect that Applied Biology and engineering technology, principle research and development in recent years substitute with tissue engineering bone/cartilage.Along with the tissue engineering technique development, tissue engineered bone cartilage is because having the structure of seamless transitional areas between similar bone, cartilage and bone cartilage simultaneously, simulation makes up the advantages such as bone cartilage structure of nature fully, has caused the concern of Chinese scholars gradually, is with a wide range of applications.Thereby, strengthen the applied basic research of tissue engineered bone cartilage, illustrate its in the cartilaginous tissue reparation effect and lapse to mechanism, for the understanding that improves tissue engineered bone cartilage, expand its range of application and have important scientific meaning.
(poly (lactic-co-glycolic acid) PLGA), by the FDA authentication, is formally included into American Pharmacopeia as pharmaceutic adjuvant in the U.S. to polylactic acid/polyglycolic acid copolymer.This class material non-toxic, no antigen has good degradable absorbability, biological safety and mechanical strength, can regulate the degradation speed of material by the control component content, is present osteochondral tissue engineering research and the class material that is most widely used.Yet because the PLGA material surface lacks cell recognition site and hydrophilic and cellular affinity deficiency, influenced cell adhesion growth in its surface, limited its clinical practice to a great extent.In recent years, along with to constantly the carrying out of PLGA study on the modification, its performance is continued to optimize, and some novel PLGA materials occur in succession.For example: the 1. PLGA biomimetic material of collagen modification, adopt the ion surface processing method to introduce functional group or functional chain at material surface, can improve the adhesion on timbering material surface.Adopt plasma treatment PLGA film, introduce the anti-base of gel of cationization, effectively improved the affinity of cell to PLGA degraded timbering material.A kind of by the nano composite membrane that HA/ collagen/the PLGA three-decker is formed, significantly improved the biological activity of material.2. arginine-glycine-aspartic acid (RGD polypeptide) modification PLGA is attached to the PLGA microsphere surface with cross-linking agent with RGD and carries out modification, shows the adhesion and the cell growth rate that improve cell.3. (hydroxyapatite, HA) modification PLGA prepare apatite/PLGA to hydroxyapatite, and the PLGA/HA composite of porous has toughness preferably, improve the synosteosis ability.
Present tissue engineering bracket based on the PLGA microsphere, owing to have advantages such as preparing easy, biodegradation is controlled and catabolite toxicity is low, slow controlled release, and be subjected to the favor of researcher.Yet the quality and quantity that forms newborn cartilaginous tissue based on the tissue engineered bone cartilage of PLGA microsphere in repairing cartilage defect all meets clinical needs far away, main cause is PLGA microsphere size heterogeneity, adhesiveness is low, the porosity heterogeneity, most cells is only docile and obedient at material surface, can't grow into to the material deep, lacks the cell seeding of capacity, and can't obtain sufficient extracellular matrix structure, limited PLGA microsphere support tissue engineering bone/cartilage to a great extent in Clinical Application.
Stem cell is one of focus of tissue engineered bone research as seed cell always.Embryonic stem cell (Embryonic stem cells, ESCs) be myeloid-lymphoid stem cell, have still have after infinite multiplication and the external long-term cultivation can induce generation from trophoderm to interior,, the ability of ectoderm all cells, but owing to reasons such as ethics and teratogenesis incidence rate be higher make the tissue engineered bone research based on ESCs relatively lag behind.Mescenchymal stem cell (Mesenchymal stem cells, MSCs) can keep undifferentiated state and have the potential of mesoderm tissues such as differentiation skeletonization, cartilage, fat, tendon, muscle, corium and bone marrow matrix at in-vitro multiplication, in a plurality of histoorgans of body, successfully extract the MSCs with differentiation potential at present.In the MSCs of multi-source, with the more research that is applied to tissue engineered bone cartilage of human marrow mescenchymal stem cell (hBMSC), these are studied prompting: hBMSC and will be with a wide range of applications aspect tissue engineered bone cartilage again.
Summary of the invention
The object of the present invention is to provide a kind of somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer to discharge microsphere support, this microsphere support microsphere bridging is even, surface texture is complete, obviously dissolving does not take place, keep desirable porosity, can form network structure, the PLGA microsphere support has TGF-β 3/BMP-2 gradient release characteristic simultaneously.
Another object of the present invention is to provide above-mentioned and discharges the preparation method of microsphere support based on the somatomedin gradient of polylactic acid-glycolic guanidine-acetic acid copolymer, and this preparation method technology is simple, be easy to control.
A further object of the invention is to provide above-mentioned somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer to discharge the application of microsphere support in the biological repair composite material of preparation bone cartilage.
First purpose of the present invention is achieved by the following technical solution: a kind of somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer discharges microsphere support, at first prepare PLGA homogenizing microsphere and TGF-β 3 and BMP-2/PLGA microsphere, lyophilizing is standby respectively, the above-mentioned microsphere of lyophilizing is scattered in makes suspension among the ddH2O, then this suspension is injected the mould that the bottom is provided with defecator, adopt ddH2O to filter and the position of adjustment ddH2O in mould, above-mentioned microsphere is piled up in mould, adopt the above-mentioned microsphere of dehydrated alcohol sintering, the somatomedin gradient release microsphere support that namely forms based on polylactic acid-glycolic guanidine-acetic acid copolymer is that TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support.
Wherein PLGA is polylactic acid/polyglycolic acid copolymer (poly (lactic-co-glycolic acid), PLGA), TGF-β 3 is transforming growth factor-beta 3 (transforming growth factor β 3, TGF-β 3), BMP-2 is bone morphogenesis protein-2 (bone morphogenetic protein-2, BMP-2), ddH
2O is distilled water.
PLGA homogenizing microsphere of the present invention prepares by the following method: be 1:0.3-3 in molar ratio, extracting lactic acid and hydroxyacetic acid polymerization at random generate PLGA, PLGA is dissolved in formation PLGA lysate among the dichloromethane DCM, the PLGA lysate is formed the homogeneous polymers drop through homogenizing, behind this polymer drop and PVAC polyvinylalcohol mixing, through stirring, form PLGA homogenizing microsphere after filtration, washing and the lyophilizing, storing for future use gets final product.
Wherein the quality percentage composition of PLGA is 1-5% in the PLGA lysate, the quality percentage composition of PVA is 0.1-1.0% in the mixed solution that forms behind polymer drop and the PVAC polyvinylalcohol mixing, mixing time is 3-4h, and freeze-drying time is 45-50h, and storage temperature is-20 ℃.
Adopt the inventive method to prepare the PLGA microsphere and have good gradient release performance, degradation rate, biocompatibility, be a kind of microsphere of homogeneous diameter simultaneously, can significantly improve mechanical property, the multiple functional Biological somatomedin of while portability, mescenchymal stem cell has good skeletonization and cartilage differentiation capability at the PLGA support.
Adopt the PLGA microsphere of the inventive method preparation, microsphere size homogeneous is moderate, support voidage homogeneous, along with the degraded of PLGA microsphere forms little and the hole of homogeneous, and then can form the space structure that is similar to spongy bone, can be the chemical environment that the bone regenerating bone or cartilage is rebuild provides physical support and the best, follow seed cell to discharge into internal stent, more help later stage area of new bone cartilaginous tissue to form.
TGF-β 3 of the present invention and BMP-2/PLGA microsphere prepare by the following method: TGF-β 3 is dissolved in makes TGF-β 3 stock solutions among the bovine serum albumin BSA, BMP-2 is dissolved in makes BMP-2 stock solution among the bovine serum albumin BSA, PLGA is dissolved in formation PLGA lysate among the dichloromethane DCM, with TGF-β 3 stock solutions, BMP-2 stock solution and PLGA lysate mixing, form uniform emulsion on ice after the ultrasonic agitation, this emulsion is formed the homogeneous polymers drop through homogenizing, behind this polymer drop and PVAC polyvinylalcohol mixing, through stirring, filter, form TGF-β 3/BMP-2-PLGA microsphere after washing and the lyophilizing, storing for future use gets final product.
The TGF-β 3 and the BMP-2/PLGA microsphere that adopt the inventive method to prepare, wherein TGF-β 3 and BMP-2 somatomedin are as the growth inducing factor of seed cell, can promote seed cell to skeletonization and the differentiation of chondrocyte direction, be conducive to more germinal cell and repair cell and assemble to damage field, be conducive to the diffusion of nutrient substance and refuse.This TGF-β 3 and BMP-2/PLGA microsphere safety non-toxic, have excellent biological compatibility, biological degradability, have good encystation and the performance of film forming.
Wherein the ultimate density of TGF-β 3 stock solutions is 1-5 μ g/mL, the ultimate density of BMP-2 stock solution is 0.1-0.5 μ g/mL, the quality percentage composition of PLGA is 1-5% in the lysate of PLGA, the volume ratio of TGF-β 3 stock solutions, BMP-2 stock solution and PLGA lysate is 1:1:20, time during ultrasonic agitation is 10-30 second on ice, and vibration amplitude is 30-60%; The quality percentage composition of PVA is 0.3-1% in the solution that forms behind polymer drop and the PVAC polyvinylalcohol mixing, and mixing time is 3-4h, and freeze-drying time is 45-50h, and storage temperature is-20 ℃.
The particle diameter of PLGA homogenizing microsphere of the present invention and TGF-β 3 and BMP-2/PLGA microsphere is 50-300 μ m.
The mass ratio 1:1 of PLGA homogenizing microsphere of the present invention and TGF-β 3 and BMP-2/PLGA microsphere.
The present invention adopts control syringe pump able to programme that this suspension is injected the mould that the bottom is provided with defecator, and described mould is cylindrical glass container, uses infusion pump and vacuum syringe pump to make ddH
2The position of O in cylindrical glass container is the 1/3-2/3 that is full of the cylindrical glass container bottom, adopt dehydrated alcohol sintering PLGA homogenizing microsphere and TGF-β 3 and BMP-2/PLGA microsphere 0.5-1.5h, formation is that TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support based on the somatomedin gradient release microsphere support of polylactic acid-glycolic guanidine-acetic acid copolymer, after the lyophilization, get final product-20 ℃ of storages.
Second purpose of the present invention is achieved by the following technical solution: above-mentioned somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer discharges the preparation method of microsphere support, contains following steps:
(1) preparation PLGA homogenizing microsphere, lyophilizing is standby;
(2) preparation TGF-β 3 and BMP-2/PLGA microsphere, lyophilizing is standby;
(3) the TGF-β 3 of lyophilizing and BMP-2/PLGA microsphere and PLGA homogenizing microsphere are scattered in ddH
2Make suspension among the O, then this suspension is injected the mould that the bottom is provided with defecator, adopt ddH
2O filters and adjusts ddH
2O is consumption in mould, in mould, pile up TGF-β 3 and BMP-2/PLGA microsphere and PLGA homogenizing microsphere, adopt dehydrated alcohol sintering TGF-β 3 and BMP-2/PLGA microsphere and PLGA homogenizing microsphere, the somatomedin gradient release microsphere support that forms based on polylactic acid-glycolic guanidine-acetic acid copolymer is that TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support.
The 3rd purpose of the present invention is achieved by the following technical solution: above-mentioned somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer discharges the application of microsphere support in the biological repair composite material of preparation bone cartilage.
Compared with prior art, the present invention has following advantage:
(1) raw material of the present invention's employing is the polymeric system of safety non-toxic, PLGA is polymerized at random by two kinds of monomer-lactic acid and hydroxyacetic acid, be a kind of degradable functional polymer organic compound, be widely used in pharmacy, medical engineering material and modernized industrial field.PLGA authenticates by FDA in the U.S., is formally included into American Pharmacopeia as pharmaceutic adjuvant.
(2) among the TGF-β 3 and BMP-2/PLGA microsphere of the present invention's preparation, TGF-β 3 and BMP-2 are as the growth inducing factor of seed cell, can promote seed cell to skeletonization and the differentiation of chondrocyte direction, be conducive to more germinal cell and repair cell and assemble to damage field.
(3) TGF-β 3 and the BMP-2/PLGA microsphere support of the present invention's preparation have certain mechanics of materials intensity, make the application of PLGA microsphere support in repairing the district's osteochondral defect that bears a heavy burden become possibility, novel synthetic technology prepares the PLGA microsphere support can reduce microsphere surface degeneration generation, be conducive to a large amount of seed cells and enter internal stent, the space structure that is similar to spongy bone is conducive to the diffusion of nutrient substance and refuse, along with the degraded of PLGA microsphere forms little and the hole of homogeneous, can be the bone regenerating bone or cartilage and rebuild physical support and best chemical environment are provided, help later stage area of new bone cartilaginous tissue to form, closely contact with surrounding tissue, easy operating, reduce wound to greatest extent, reduce operating difficulty, reduce patient's misery, reduce risk of infection, cicatrization and medical expense.
(4) the present invention prepares in the microsphere support, skeletonization and cartilage-inducing factor (BMP-2 and TGF-β 3) discharge with the Cmax gradient along the microsphere support two ends to offside respectively, seed cell forms class cartilage and analogy osteoblast along somatomedin Concentraton gradient induction in the PLGA support, and obtain the bone of similar natural structure, cartilage and seamless transitional areas, can be osseocartilaginous regeneration and reconstruction best spatial environments is provided, rebuild the physicochemical environment that provides best for the bone regenerating bone or cartilage, the extracellular matrix components of simulation natural bone cartilaginous tissue, make material have excellent biocompatibility and adjustable physical and mechanical properties, biodegradability more helps later stage area of new bone cartilage to form.
(5) the novel gradient release PLGA microsphere support of the present invention's preparation has not only kept the original good biocompatibility of PLGA, but also significantly improved mechanical property, the effect of bone chondrocyte induction of PLGA microsphere support and carried the seed cell ability, has excellent biocompatibility, the seed cell of portability q.s is for the treatment of the osteochondral defect that is caused by multiple diseases such as tumor, wound, severe infections, congenital malformations.
(6) preparation technology of the present invention is easy to control, easy and simple to handle, adopt the inventive method to prepare microsphere support and can reduce wound to greatest extent, reduce operating difficulty, reduce patient's misery, reduce risk of infection, cicatrization and medical expense, have certain advance and novelty in the tissue engineered bone cartilage research field, can be tissue engineered bone cartilage research and clinical practice new approaches and new method are provided, this timbering material can be realized industrialization production, Related product has the bigger market competitiveness, can be applied to well in all kinds of reparations based on cartilage defect, it is good to give full play to it, the repair ability of nature has wide clinical practice and market prospect.
Description of drawings
Fig. 1 is that the SEM figure of the support of the microsphere porous property of PLGA of sintering preparation among the embodiment 2 and human umbilical cord mesenchymal stromal cell are cultivated 2 real time imagings when all in support, sequence number 1 and 2 wherein: expression SEM shows the support of the microsphere porous property of PLGA of sintering preparation, visible typical little connection network structure among the figure of sequence number 1, scale: sequence number 1 and sequence number 2 are respectively 100 μ m and 50 μ m, sequence number 3 and 4 figure are the real time imaging of human umbilical cord mesenchymal stromal cell when cultivating for 2 weeks in support, scale: 1000 μ m;
Fig. 2 is that the support inoculating cell of the microsphere porous property of PLGA of sintering preparation among the embodiment 2 is cultivated 3 all fluorescence micrographs, and wherein sequence number 1 is represented living cells and dead cell, and the figure of sequence number 2 represents living cells, and the figure of sequence number 3 represents dead cell.Scale: 100 μ m.
Fig. 3 is that the TGF-β 3/ BMP-2 gradient that makes up among the embodiment 2 discharges PLGA microsphere support figure, and wherein sequence number 1 is represented the bone cartilage overall diagram of preparation, sequence number 2 expression micrograms.
The specific embodiment
Following examples only are used for setting forth the present invention, and protection scope of the present invention is not only to be confined to following examples.The those of ordinary skill of described technical field all can be realized purpose of the present invention according to above content disclosed by the invention and scope that each parameter is got.
1. make up homogenizing PLGA microsphere
The PLGA microsphere of preparation diameter 50 μ m.(mol ratio is the lactic acid of 1:1 to the PLGA composition: glycolic, the molecular weight of polymer ~ 25,000) makes PLGA through polymerization at random, with dichloromethane DCM(DCM 30%W/V, mass volume ratio) dissolving PLGA forms the PLGA lysate, wherein the quality percentage composition of PLGA is 3% in the PLGA lysate, this polymer solution is by ultrasonic sensor control waveform generator, through a coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, inflow contains 0.5%(quality percentage composition) in the beaker of PVA, form PLGA polymer drop.Early stage PLGA polymer drop need stir 3-4 hour, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.At last, PLGA polymer microballoon lyophilizing 48 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is standby.
2. make up TGF-β 3 and BMP-2/PLGA microsphere
100 μ g BMP-2 add 10 mL0.5 μ g/mL bovine serum albumin (BSA) at the PBS(phosphate buffer) obtain 0.2 μ g/mLBMP-2 stock solution.50 μ g TGF-β, 3 usefulness, 25 μ g/mL BSA are dissolved as TGF-β 3 stock solutions of 1 μ g/mL, and 500 mg PLGA are dissolved in 5mL DCM, and (WT6.5 restrains, 20%W/V).250 μ LBMP-2 and TGF-β 3 stock solutions are mixed with 5 mL PLGA solution respectively, and ultrasonic agitation (50% vibration amplitude, 20 seconds) forms uniform PLGA protein emulsion on ice.This polymer solution is by ultrasonic sensor control waveform generator, through a coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, it is in 0.5% the PVA beaker that inflow contains the quality percentage composition, form TGF-β 3 and BMP-2/PLGA polymer drop, earlier T GF-β 3 and BMP-2/PLGA polymer drop need stir 3-4 hour, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.Last TGF-β 3 and the lyophilizing of BMP-2/PLGA polymer microballoon 48 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is standby.TGF-β 3 and the BMP-2/PLGA microsphere of different-grain diameter collapse the flow speed control microspherulite diameter by microgranule, prepare in batches.
3. make up TGF-β 3/ BMP-2 gradient and discharge the PLGA microsphere support
Be the lyophilizing TGF-β 3 of 50 μ m and BMP-2/PLGA microsphere and blank PLGA microsphere by quality volume (g/mL) than being 2.5% to be dispersed in ddH with diameter
2Among the O, wherein the mass ratio of TGF-β 3 and BMP-2/PLGA microsphere and blank PLGA microsphere is that 1:1 is respectively charged into 20 mL syringes.Use control syringe pump able to programme that this suspension is injected cylindrical glass mould (4 millimeters of diameters).Mold bottom is equipped with filter (granule reservation〉3 μ m), ddH
2O filters, and microsphere accumulates at mould.Use an extra infusion pump and vacuum syringe pump, make distilled water keep certain level at mould, use infusion pump and vacuum syringe pump to make ddH
2The position of O in cylindrical glass container is to be full of 1/3 of cylindrical glass container bottom, piles up PLGA microsphere, TGF-β 3 and BMP-2/PLGA microsphere.Dehydrated alcohol sintering PLGA microsphere, TGF-β 3 and BMP-2/PLGA microsphere 1 hour form TGF-β 3/ BMP-2 gradient and discharge PLGA microsphere support, lyophilization 2 days ,-20 ℃ of storages.
1. make up homogenizing PLGA microsphere
The PLGA microsphere of preparation diameter 100 μ m.(mol ratio is the lactic acid of 1:0.3 to the PLGA composition: glycolic, the molecular weight of polymer ~ 25,000) makes PLGA through polymerization at random, use the DCM(dichloromethane) (DCM 30%W/V, mass volume ratio) dissolving PLGA forms the PLGA lysate, wherein the quality percentage composition of PLGA is 1% in the PLGA lysate, this polymer solution is by ultrasonic sensor control waveform generator, through a coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, inflow contains 0.8%(quality percentage composition) in the beaker of PVA, form PLGA polymer drop.Early stage PLGA polymer drop need stir 3-4 hour, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.At last, PLGA polymer microballoon lyophilizing 50 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is standby.
2. make up TGF-β 3 and BMP-2/PLGA microsphere
Get BMP-2 and add bovine serum albumin (BSA) at the PBS(phosphate buffer) obtain 0.3 μ g/mLBMP-2 stock solution.Get TGF-β 3 usefulness BSA and be dissolved as TGF-β 3 stock solutions of 2 μ g/mL, getting PLGA is dissolved in DCM to get the quality percentage composition is 2% PLGA lysate, BMP-2 stock solution and TGF-β 3 stock solutions are mixed for 1:1:20 by volume with the PLGA lysate respectively, ultrasonic agitation (50% vibration amplitude on ice, 20 seconds), form uniform PLGA protein emulsion.This polymer solution is by ultrasonic sensor control waveform generator, through a coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, it is in 0.3% the PVA beaker that inflow contains the quality percentage composition, form TGF-β 3 and BMP-2/PLGA polymer drop, earlier T GF-β 3 and BMP-2/PLGA polymer drop need stir 3-4 hour, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.Last TGF-β 3 and the lyophilizing of BMP-2/PLGA polymer microballoon 50 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is standby.
TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support and prepares by the following method:
Adopting the above-mentioned diameter for preparing 100 μ m TGF-β 3 and BMP-2/PLGA microsphere and diameter is the cylindrical structure support that the blank PLGA microsphere of 100 μ m prepares 4 mm (height) x 4 mm (diameter).Lyophilizing TGF-β 3 and BMP-2/PLGA microsphere and blank PLGA microsphere are dispersed in ddH
2O (2.5%W/V), wherein the mass ratio of TGF-β 3 and BMP-2/PLGA microsphere and blank PLGA microsphere is that 1:1 is respectively charged into 20 mL syringes.Use control syringe pump able to programme that this suspension is injected cylindrical glass mould (4 millimeters of diameters).Mold bottom is equipped with filter (granule reservation〉3 μ m), ddH
2O filters, and microsphere accumulates at mould.Use an extra infusion pump and vacuum syringe pump, make distilled water keep certain level at mould, use infusion pump and vacuum syringe pump to make ddH
2The position of O in cylindrical glass container is to be full of 2/3 of cylindrical glass container bottom, piles up PLGA microsphere, TGF-β 3 and BMP-2/PLGA microsphere.Dehydrated alcohol sintering PLGA microsphere, TGF-β 3 and BMP-2/PLGA microsphere 1 hour, forming the inner space is that hexagon TGF-β 3/ BMP-2 gradient discharges PLGA microsphere support, lyophilization 2 days ,-20 ℃ of storages.
Embodiment 3
1. make up homogenizing PLGA microsphere
The PLGA microsphere of preparation diameter 150 μ m.(mol ratio is the lactic acid of 1:2 to the PLGA composition: glycolic, the molecular weight of polymer ~ 25,000) makes PLGA through polymerization at random, use the DCM(dichloromethane) (DCM 30%W/V, mass volume ratio) dissolving PLGA forms the PLGA lysate, wherein the quality percentage composition of PLGA is 5% in the PLGA lysate, this polymer solution is by ultrasonic sensor control waveform generator, through a coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, inflow contains 1.0%(quality percentage composition) in the beaker of PVA, form PLGA polymer drop.Early stage PLGA polymer drop need stir 3-4 hour, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.At last, PLGA polymer microballoon lyophilizing 45 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is standby.
2. make up TGF-β 3 and BMP-2/PLGA microsphere
Get BMP-2 and add bovine serum albumin (BSA) at the PBS(phosphate buffer) obtain 0.4 μ g/mLBMP-2 stock solution.Get TGF-β 3 usefulness BSA and be dissolved as TGF-β 3 stock solutions of 3 μ g/mL, getting PLGA is dissolved in DCM to get the quality percentage composition is 3% PLGA lysate, BMP-2 stock solution and TGF-β 3 stock solutions are mixed for 1:1:20 by volume with the PLGA lysate respectively, ultrasonic agitation (50% vibration amplitude on ice, 30 seconds), form uniform PLGA protein emulsion.This polymer solution is by ultrasonic sensor control waveform generator, through a coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, it is in 0.8% the PVA beaker that inflow contains the quality percentage composition, form TGF-β 3 and BMP-2/PLGA polymer drop, earlier T GF-β 3 and BMP-2/PLGA polymer drop need stir 3-4 hour, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.Last TGF-β 3 and the lyophilizing of BMP-2/PLGA polymer microballoon 50 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is standby.
TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support and prepares by the following method:
Employing diameter 150 μ m TGF-β 3 and BMP-2/PLGA microsphere and diameter are the cylindrical structure support that the blank PLGA microsphere of 150 μ m prepares 4 mm (height) x 4 mm (diameter).Lyophilizing TGF-β 3 and BMP-2/PLGA microsphere and blank PLGA microsphere are dispersed in ddH
2O (2.5%W/V), wherein the mass ratio of TGF-β 3 and BMP-2/PLGA microsphere and blank PLGA microsphere is 1:1, is respectively charged into 20mL and penetrates device.Use control syringe pump able to programme that this suspension is injected cylindrical glass mould (4 millimeters of diameters).Mold bottom is equipped with filter (granule reservation〉3 μ m), ddH
2O filters, and microsphere accumulates at mould.Use an extra infusion pump and vacuum syringe pump, make distilled water keep certain level at mould, use infusion pump and vacuum syringe pump to make ddH
2The position of O in cylindrical glass container is to be full of 1/2 of cylindrical glass container bottom, piles up PLGA microsphere, TGF-β 3 and BMP-2/PLGA microsphere.Dehydrated alcohol sintering PLGA microsphere, TGF-β 3 and BMP-2/PLGA microsphere 1 hour, forming the inner space is that hexagon TGF-β 3/ BMP-2 gradient discharges PLGA microsphere support, lyophilization 2 days ,-20 ℃ of storages.
Embodiment 4
1. make up homogenizing PLGA microsphere
The PLGA microsphere of preparation diameter 200 μ m.(mol ratio is the lactic acid of 1:3 to the PLGA composition: glycolic, the molecular weight of polymer ~ 25,000) makes PLGA through polymerization at random, use the DCM(dichloromethane) (DCM 30%W/V, mass volume ratio) dissolving PLGA forms the PLGA lysate, wherein the quality percentage composition of PLGA is 2% in the PLGA lysate, this polymer solution is by ultrasonic sensor control waveform generator, through a coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, inflow contains 0.3%(quality percentage composition) in the beaker of PVA, form PLGA polymer drop.Early stage PLGA polymer drop need stir 3-4 hour, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.At last, PLGA polymer microballoon lyophilizing 47 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is standby.
2. make up TGF-β 3 and BMP-2/PLGA microsphere
Get BMP-2 and add bovine serum albumin (BSA) at the PBS(phosphate buffer) obtain 0.5 μ g/mLBMP-2 stock solution.Get TGF-β 3 usefulness BSA and be dissolved as TGF-β 3 stock solutions of 5 μ g/mL, getting PLGA is dissolved in DCM to get the quality percentage composition is 5% PLGA lysate, BMP-2 stock solution and TGF-β 3 stock solutions are mixed for 1:1:20 by volume with the PLGA lysate respectively, ultrasonic agitation (60% vibration amplitude on ice, 10 seconds), form uniform PLGA protein emulsion.This polymer solution is by ultrasonic sensor control waveform generator, through a coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, it is in 1.0% the PVA beaker that inflow contains the quality percentage composition, form TGF-β 3 and BMP-2/PLGA polymer drop, earlier T GF-β 3 and BMP-2/PLGA polymer drop need stir 3-4 hour, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.Last TGF-β 3 and the lyophilizing of BMP-2/PLGA polymer microballoon 47 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is standby.
TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support and prepares by the following method:
Employing diameter 200 μ m TGF-β 3 and BMP-2/PLGA microsphere and diameter are the cylindrical structure support that the blank PLGA microsphere of 200 μ m prepares 4 mm (height) x 4 mm (diameter).Lyophilizing TGF-β 3 and BMP-2/PLGA microsphere and blank PLGA microsphere are dispersed in ddH
2O (2.5% W/ V), wherein the mass ratio of TGF-β 3 and BMP-2/PLGA microsphere and blank PLGA microsphere is 1:1, is respectively charged into 20 mL and penetrates device.Use control syringe pump able to programme that this suspension is injected cylindrical glass mould (4 millimeters of diameters).Mold bottom is equipped with filter (granule reservation〉3 μ m), ddH
2O filters, and microsphere accumulates at mould.Use an extra infusion pump and vacuum syringe pump, make distilled water keep certain level at mould, use infusion pump and vacuum syringe pump to make ddH
2The position of O in cylindrical glass container is to be full of 1/2 of cylindrical glass container bottom, piles up PLGA microsphere, TGF-β 3 and BMP-2/PLGA microsphere.100% ethanol sintering PLGA microsphere 1 hour, forming the inner space is that hexagon TGF-β 3/ BMP-2 gradient discharges PLGA microsphere support, lyophilization 2 days ,-20 ℃ of storages.
Embodiment 5
1. make up homogenizing PLGA microsphere
The PLGA microsphere of preparation diameter 250 μ m.(mol ratio is the lactic acid of 1:0.5 to the PLGA composition: glycolic, the molecular weight of polymer ~ 25,000) makes PLGA through polymerization at random, use the DCM(dichloromethane) (DCM 30%W/V, mass volume ratio) dissolving PLGA forms the PLGA lysate, wherein the quality percentage composition of PLGA is 2.5% in the PLGA lysate, this polymer solution is by ultrasonic sensor control waveform generator, through a coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, inflow contains 0.6%(quality percentage composition) in the beaker of PVA, form PLGA polymer drop.Early stage PLGA polymer drop need stir 3-4 hour, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.At last, PLGA polymer microballoon lyophilizing 49 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is standby.The homogenizing PLGA microsphere of different-grain diameter collapses the flow speed control microspherulite diameter by microgranule, prepares in batches.
2. make up TGF-β 3 and BMP-2/PLGA microsphere
Get BMP-2 and add bovine serum albumin (BSA) at the PBS(phosphate buffer) obtain 0.35 μ g/mLBMP-2 stock solution.Get TGF-β 3 usefulness BSA and be dissolved as TGF-β 3 stock solutions of 4.5 μ g/mL, getting PLGA is dissolved in DCM to get the quality percentage composition is 3.5% PLGA lysate, BMP-2 stock solution and TGF-β 3 stock solutions are mixed for 1:1:20 by volume with the PLGA lysate respectively, ultrasonic agitation (50% vibration amplitude on ice, 30 seconds), form uniform PLGA protein emulsion.This polymer solution is by ultrasonic sensor control waveform generator, through a coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, it is in 0.5% the PVA beaker that inflow contains the quality percentage composition, form TGF-β 3 and BMP-2/PLGA polymer drop, earlier T GF-β 3 and BMP-2/PLGA polymer drop need stir 3-4 hour, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.Last TGF-β 3 and the lyophilizing of BMP-2/PLGA polymer microballoon 49 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is standby.
TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support and prepares by the following method:
Employing diameter 250 μ m TGF-β 3 and BMP-2/PLGA microsphere and diameter are the cylindrical structure support that the blank PLGA microsphere of 250 μ m prepares 4mm (height) x4mm (diameter).Lyophilizing TGF-β 3 and BMP-2/PLGA microsphere and blank PLGA microsphere are dispersed in ddH
2O(2.5%W/V, the mass volume ratio of microsphere and water), wherein the mass ratio of TGF-β 3 and BMP-2/PLGA microsphere and blank PLGA microsphere is 1:1, is respectively charged into 20 mL and penetrates device.Use control syringe pump able to programme that this suspension is injected cylindrical glass mould (4 millimeters of diameters).Mold bottom is equipped with filter (granule reservation〉3 μ m), ddH
2O filters, and microsphere accumulates at mould.Use an extra infusion pump and vacuum syringe pump, make distilled water keep certain level at mould, use infusion pump and vacuum syringe pump to make ddH
2The position of O in cylindrical glass container is to be full of 1/2 of cylindrical glass container bottom, piles up PLGA microsphere, TGF-β 3 and BMP-2/PLGA microsphere.Dehydrated alcohol sintering PLGA microsphere,, TGF-β 3 and BMP-2/PLGA microsphere 1 hour, forming the inner space is that hexagon TGF-β 3/ BMP-2 gradient discharges PLGA microsphere support, lyophilization 2 days ,-20 ℃ of storages.
Embodiment 6
1. make up homogenizing PLGA microsphere
The PLGA microsphere of preparation diameter 300 μ m.(mol ratio is the lactic acid of 1:1 to the PLGA composition: glycolic, the molecular weight of polymer ~ 25,000) makes PLGA through polymerization at random, use the DCM(dichloromethane) (DCM 30%W/V, mass volume ratio) dissolving PLGA forms the PLGA lysate, wherein the quality percentage composition of PLGA is 5% in the PLGA lysate, this polymer solution is by ultrasonic sensor control waveform generator, through a coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, inflow contains 0.5%(quality percentage composition) in the beaker of PVA, form PLGA polymer drop.Early stage PLGA polymer drop need stir 3-4 hour, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.At last, PLGA polymer microballoon lyophilizing 48 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is standby.The homogenizing PLGA microsphere of different-grain diameter collapses the flow speed control microspherulite diameter by microgranule, prepares in batches.
2. make up TGF-β 3 and BMP-2/PLGA microsphere
Get BMP-2 and add bovine serum albumin (BSA) at the PBS(phosphate buffer) obtain 0.1 μ g/mLBMP-2 stock solution.Get TGF-β 3 usefulness BSA and be dissolved as TGF-β 3 stock solutions of 1 μ g/mL, getting PLGA is dissolved in DCM to get the quality percentage composition is 1% PLGA lysate, BMP-2 stock solution and TGF-β 3 stock solutions are mixed for 1:1:20 by volume with the PLGA lysate respectively, ultrasonic agitation (50% vibration amplitude on ice, 30 seconds), form uniform PLGA protein emulsion.This polymer solution is by ultrasonic sensor control waveform generator, through a coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, it is in 0.5% the PVA beaker that inflow contains the quality percentage composition, form TGF-β 3 and BMP-2/PLGA polymer drop, earlier T GF-β 3 and BMP-2/PLGA polymer drop need stir 3-4 hour, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.Last TGF-β 3 and the lyophilizing of BMP-2/PLGA polymer microballoon 48 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is standby.
TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support and prepares by the following method:
Employing diameter 300 μ m TGF-β 3 and BMP-2/PLGA microsphere and diameter are the cylindrical structure support that the blank PLGA microsphere of 300 μ m prepares 4mm (height) x4mm (diameter).Lyophilizing TGF-β 3 and BMP-2/PLGA microsphere and blank PLGA microsphere are dispersed in ddH
2O(2.5%W/V, the mass volume ratio of microsphere and water), wherein the mass ratio of TGF-β 3 and BMP-2/PLGA microsphere and blank PLGA microsphere is 1:1, is respectively charged into 20 mL and penetrates device.Use control syringe pump able to programme that this suspension is injected cylindrical glass mould (4 millimeters of diameters).Mold bottom is equipped with filter (granule reservation〉3 μ m), ddH
2O filters, and microsphere accumulates at mould.Use an extra infusion pump and vacuum syringe pump, make distilled water keep certain level at mould, use infusion pump and vacuum syringe pump to make ddH
2The position of O in cylindrical glass container is to be full of 1/2 of cylindrical glass container bottom, piles up PLGA microsphere, TGF-β 3 and BMP-2/PLGA microsphere.Dehydrated alcohol sintering PLGA microsphere,, TGF-β 3 and BMP-2/PLGA microsphere 1 hour, forming the inner space is that hexagon TGF-β 3/ BMP-2 gradient discharges PLGA microsphere support, lyophilization 2 days ,-20 ℃ of storages.
Below the TGF-β 3/ BMP-2 gradient that makes up for above-described embodiment discharges the application test of PLGA microsphere support:
1.hBMSCs cultivate
In vitro culture and osteogenic induction from body hBMSCs are gone up sour jujube through puncture extraction bone marrow 10-20 mL behind patient's bone, the bone marrow that obtains is placed (density 1.073 g/L on the Percoll separating medium, Pharmacia company, the U.S.), the ratio of bone marrow and separating medium is 1: 2, centrifugal 30 min of 2 550 r/min, cloud cellular layer in the middle of drawing is with 2 * 10
7Cell/cm
2Density be inoculated in culture dish, carry out cell in vitro amplification, it is standby to get the 4th generation hBMSCs.
Table 1 experiment grouping amounts to 10 groups
| Factor | Levels |
| Microsphere diameter | 50、100、150、200、250、300μm |
| Somatomedin | TGF-β3, BMP-2 |
Below be the application test of the TGF-β 3/ BMP-2 gradient release PLGA microsphere support of structure among the embodiment 2:
Handle through sterilization, 50-100 μ LhUCMSCs cell suspension adds the TGF-β 3/ BMP-2 gradient for preparing among the embodiment 2 and discharges PLGA microsphere support, the about 25 million cells/mL of final cell concentration.Behind the inoculating cell 6 hours, add culture fluid, changed liquid once, and cultivated for 6 weeks altogether in per two days.Culture fluid comprises: DMEM-high glucose, 10% calf serum (FBS), 25 μ g/mL ascorbic acid (ascorbic acid), (non-essential amino acids is NEAA) with 1% antibiotic (antibiotics) for 1% non essential amino acid.It is standby to insert the cell incubator, condition of culture: 37 ℃ of temperature, CO
2Concentration 90%, result such as Fig. 1 are shown in Fig. 2 and Fig. 3.
Above-described embodiment is preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included in protection scope of the present invention.
Claims (10)
1. the somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer discharges microsphere support, it is characterized in that preparing by the following method: at first prepare PLGA homogenizing microsphere and TGF-β 3 and BMP-2/PLGA microsphere, respectively lyophilizing is standby, and the above-mentioned microsphere of lyophilizing is scattered in ddH
2Make suspension among the O, then this suspension is injected the mould that the bottom is provided with defecator, adopt ddH
2O filters and adjusts ddH
2The position of O in mould, above-mentioned microsphere is piled up in mould, adopts the above-mentioned microsphere of dehydrated alcohol sintering, and the somatomedin gradient release microsphere support that namely forms based on polylactic acid-glycolic guanidine-acetic acid copolymer is that TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support.
2. the somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer according to claim 1 discharges microsphere support, it is characterized in that described PLGA homogenizing microsphere prepares by the following method: be 1:0.3-3 in molar ratio, extracting lactic acid and hydroxyacetic acid polymerization at random generate PLGA, PLGA is dissolved in formation PLGA lysate among the dichloromethane DCM, the PLGA lysate is formed the homogeneous polymers drop through homogenizing, behind this polymer drop and PVAC polyvinylalcohol mixing, through stirring, form PLGA homogenizing microsphere after filtration, washing and the lyophilizing, storing for future use gets final product.
3. the somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer according to claim 2 discharges microsphere support, it is characterized in that: wherein the quality percentage composition of PLGA is 1-5% in the PLGA lysate, the quality percentage composition of PVA is 0.1-1.0% in the mixed solution that forms behind polymer drop and the PVAC polyvinylalcohol mixing, mixing time is 3-4h, freeze-drying time is 45-50h, and storage temperature is-20 ℃.
4. the somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer according to claim 1 discharges microsphere support, it is characterized in that described TGF-β 3 and BMP-2/PLGA microsphere prepare by the following method: TGF-β 3 is dissolved in makes TGF-β 3 stock solutions among the bovine serum albumin BSA, BMP-2 is dissolved in makes BMP-2 stock solution among the bovine serum albumin BSA, PLGA is dissolved in formation PLGA lysate among the dichloromethane DCM, with TGF-β 3 stock solutions, BMP-2 stock solution and PLGA lysate mixing, form uniform emulsion on ice after the ultrasonic agitation, this emulsion is formed the homogeneous polymers drop through homogenizing, behind this polymer drop and PVAC polyvinylalcohol mixing, through stirring, filter, form TGF-β 3/BMP-2-PLGA microsphere after washing and the lyophilizing, storing for future use gets final product.
5. the somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer according to claim 4 discharges microsphere support, it is characterized in that: wherein the ultimate density of TGF-β 3 stock solutions is 1-5 μ g/mL, the ultimate density of BMP-2 stock solution is 0.1-0.5 μ g/mL, the quality percentage composition of PLGA is 1-5% in the lysate of PLGA, the volume ratio of TGF-β 3 stock solutions, BMP-2 stock solution and PLGA lysate is 1:1:20, time during ultrasonic agitation is 10-30 second on ice, and vibration amplitude is 30-60%; The quality percentage composition of PVA is 0.3-1% in the solution that forms behind polymer drop and the PVAC polyvinylalcohol mixing, and mixing time is 3-4h, and freeze-drying time is 45-50h, and storage temperature is-20 ℃.
6. the somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer according to claim 1 discharges microsphere support, and it is characterized in that: the particle diameter of described PLGA homogenizing microsphere and TGF-β 3 and BMP-2/PLGA microsphere is 50-300 μ m.
7. the somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer according to claim 1 discharges microsphere support, it is characterized in that: the mass ratio 1:1 of PLGA homogenizing microsphere and TGF-β 3 and BMP-2/PLGA microsphere.
8. the somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer according to claim 1 discharges microsphere support, it is characterized in that: adopt control syringe pump able to programme that this suspension is injected the mould that the bottom is provided with defecator, described mould is cylindrical glass container, uses infusion pump and vacuum syringe pump to make ddH
2The position of O in cylindrical glass container is the 1/3-2/3 that is full of the cylindrical glass container bottom, adopt dehydrated alcohol sintering PLGA homogenizing microsphere and TGF-β 3 and BMP-2/PLGA microsphere 0.5-1.5h, formation is that TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support based on the somatomedin gradient release microsphere support of polylactic acid-glycolic guanidine-acetic acid copolymer, after the lyophilization, get final product-20 ℃ of storages.
9. each described somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer of claim 1-8 discharges the preparation method of microsphere support, it is characterized in that comprising following steps:
(1) preparation PLGA homogenizing microsphere, lyophilizing is standby;
(2) preparation TGF-β 3 and BMP-2/PLGA microsphere, lyophilizing is standby;
(3) the TGF-β 3 of lyophilizing and BMP-2/PLGA microsphere and PLGA homogenizing microsphere are scattered in ddH
2Make suspension among the O, then this suspension is injected the mould that the bottom is provided with defecator, adopt ddH
2O filters and adjusts ddH
2O is consumption in mould, in mould, pile up TGF-β 3 and BMP-2/PLGA microsphere and PLGA homogenizing microsphere, adopt dehydrated alcohol sintering TGF-β 3 and BMP-2/PLGA microsphere and PLGA homogenizing microsphere, the somatomedin gradient release microsphere support that forms based on polylactic acid-glycolic guanidine-acetic acid copolymer is that TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support.
10. each described somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer of claim 1-8 discharges the application of microsphere support in the biological repair composite material of preparation bone cartilage.
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| JP5890222B2 (en) * | 2012-03-30 | 2016-03-22 | 株式会社ジーシー | Cultured cartilage tissue material |
| CN103550824B (en) * | 2013-11-01 | 2016-03-02 | 南京医科大学附属口腔医院 | A kind of preparation method of support of inducting osseous tissue regeneration |
| CN104096263B (en) * | 2014-06-30 | 2015-07-15 | 北京大学口腔医院 | Submicron nucleocapsid microsphere material capable of releasing active factors on time and preparation method of submicron nucleocapsid microsphere material |
| CN104689323A (en) * | 2015-01-23 | 2015-06-10 | 上海大学 | Oil-soluble drug microsphere with slow release function and preparation method thereof |
| CN107185044A (en) * | 2017-05-22 | 2017-09-22 | 北京大学第三医院 | A kind of cartilage tissue engineered recovery support and preparation method thereof |
| CN108619491B (en) * | 2018-03-26 | 2020-08-18 | 杭州市萧山区中医院 | Application of PLGA micro-capsule scaffold encapsulated with BMP-2and PRF (platelet-derived growth factor) in preparation of medicine for promoting healing of aponeurosis |
| CN110169959B (en) * | 2019-05-20 | 2022-11-15 | 中国人民解放军总医院 | Growth factor slow-release microsphere, tissue engineering cartilage composite scaffold and preparation method |
| CN110403736A (en) * | 2019-08-09 | 2019-11-05 | 常州市第二人民医院 | A kind of super porous titanium alloy of 3D printing facilitates bone surface method of modifying |
| CN114948906B (en) * | 2022-04-13 | 2023-06-30 | 南京医科大学附属口腔医院 | Preparation method and application of controllable slow-release polylactic acid-glycolic acid copolymer microcapsule |
| CN115040697A (en) * | 2022-04-25 | 2022-09-13 | 中国人民解放军空军军医大学 | Hydrogel for accelerating regeneration of tracheal epithelium and cartilage, preparation method and bracket |
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| CN101209240A (en) * | 2007-12-21 | 2008-07-02 | 东华大学 | Medicine gradient zero level implantation controlled-release drug administration device and preparation thereof |
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