CN102319449A - 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 the biological repair composite material of a kind of bone cartilage, be specifically related to a kind of somatomedin gradient and discharge microsphere support based on polylactic acid-glycolic guanidine-acetic acid copolymer.
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 efficacious therapy method.But the operating substitution joint not only costs an arm and a leg, and has complication dangerous.Applied Biology and engineering technology, Research on Principle exploitation in recent years substitutes and uses tissue engineering bone/cartilage to open up new way and new method as the reparation of cartilage defect.Along with the tissue engineering technique development; Tissue engineered bone cartilage is because of have 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), through the FDA authentication, is formally included into American Pharmacopeia as pharmaceutic adjuvant in the U.S. to polylactic acid/polyglycolic acid copolymer.This type material non-toxic, no antigen has good degradable absorbability, biological safety and mechanical strength, can regulate the degradation speed of material through the control component content, is present osteochondral tissue engineering research and application one type of material the most widely.Yet because the PLGA material surface lacks the cell recognition site and hydrophilic is not enough with cellular affinity, influenced cell adhesion in its surface and grown, largely limit its clinical practice.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 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 porous PLGA/HA composite 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 receive the favor of researcher.Yet the quality and quantity that in repairing cartilage defect, forms newborn cartilaginous tissue based on the tissue engineered bone cartilage of PLGA microsphere all meets clinical needs far away, and main cause is a PLGA microsphere size heterogeneity, and 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 competent extracellular matrix structure, largely limit PLGA microsphere support tissue engineering bone/cartilage 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 MSCs at present with differentiation potential.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 aspect tissue engineered bone cartilage, be with a wide range of applications again.
Summary of the invention
The object of the present invention is to provide a kind of somatomedin gradient to discharge microsphere support based on polylactic acid-glycolic guanidine-acetic acid copolymer; This microsphere support microsphere bridging is even; Surface texture is complete, and obviously dissolving does not take place, and keeps desirable porosity; Can form RF, 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 method for preparing of microsphere support based on the somatomedin gradient of polylactic acid-glycolic guanidine-acetic acid copolymer, and this method for preparing 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 realizes through following technical scheme: 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 subsequent use respectively; Freeze dried above-mentioned microsphere is scattered in processes 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 promptly 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), and BMP-2 is bone morphogenesis protein-2 (bone morphogenetic protein-2; BMP-2), ddH
2O is a distilled water.
PLGA homogenizing microsphere of the present invention prepares through following method: be 1:0.3-3 in molar ratio; Extracting lactic acid and hydroxyacetic acid polymerization at random generate PLGA, PLGA are dissolved in form the PLGA lysate among the dichloromethane DCM, and 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 on the PLGA support.
Adopt the PLGA microsphere of the inventive method preparation; Microsphere size homogeneous is moderate, and support voidage homogeneous is 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 prepares through following method with the BMP-2/PLGA microsphere: TGF-β 3 is dissolved in processes TGF-β 3 stock solutions among the bovine serum albumin BSA; BMP-2 is dissolved in processes BMP-2 stock solution among the bovine serum albumin BSA, PLGA is dissolved in forms the 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; Form TGF-β 3/BMP-2-PLGA microsphere after filtration, washing and the lyophilizing, storing for future use gets final product.
The TGF-β 3 and 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; Help more germinal cell and repair cell and assemble, help the diffusion of nutrient substance and refuse to damage field.This TGF-β 3 with BMP-2/PLGA microsphere safety non-toxic, have excellent biological compatibility, biological degradability, have good encystation and film forming performance.
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, and the 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 a 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 realizes through following technical scheme: above-mentioned somatomedin gradient based on polylactic acid-glycolic guanidine-acetic acid copolymer discharges the method for preparing of microsphere support, contains following steps:
(1) preparation PLGA homogenizing microsphere, lyophilizing is subsequent use;
(2) preparation TGF-β 3 and BMP-2/PLGA microsphere, lyophilizing is subsequent use;
(3) freeze dried TGF-β 3 is scattered in ddH with BMP-2/PLGA microsphere and PLGA homogenizing microsphere
2Process suspension among the O, then this suspension is injected the mould that the bottom is provided with defecator, adopt ddH
2O filters and adjustment 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 realizes through following technical scheme: 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 by two kinds of monomer-lactic acid and hydroxyacetic acid at random; Be a kind of degradable functional polymer organic compound, be widely used in pharmacy, medical engineering material and modernized industrial field.Through the FDA authentication, formally included into American Pharmacopeia at U.S. PLGA as pharmaceutic adjuvant.
(2) among the TGF-β 3 and BMP-2/PLGA microsphere of the present invention's preparation; The TGF-β 3 and the growth inducing factor of BMP-2 as seed cell; Can promote seed cell to skeletonization and the differentiation of chondrocyte direction, help more germinal cell and repair cell and assemble to damage field.
(3) the TGF-β 3 of the present invention's preparation has certain mechanics of materials intensity with the BMP-2/PLGA microsphere support; 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, helps a large amount of seed cells and gets into internal stents, and the space structure that is similar to spongy bone helps 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 physical support and best chemical environment are provided, help later stage area of new bone cartilaginous tissue to form, closely contact with surrounding tissue with rebuilding; Easy operating, reduce wound to greatest extent, reduce operating difficulty, reduce less patient suffering, 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 to offside along the microsphere support two ends respectively; Seed cell forms type cartilage and analogy osteoblast along somatomedin Concentraton gradient induction in the PLGA support; And obtain bone, cartilage and the seamless transitional areas of similar natural structure; Can be osseocartilaginous regeneration and reconstruction best spatial environments is provided, for the bone regenerating bone or cartilage is rebuild the physicochemical environment that provides best, the extracellular matrix components of simulation nature bone cartilaginous tissue; Make material have excellent biocompatibility and adjustable physical and mechanical properties, biodegradability, more help 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, the treatment of the osteochondral defect that is used for causing 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 less patient suffering, 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, and Related product has the bigger market competitiveness, can be applied to well in all kinds of reparations based on cartilage defect; Give full play to its good, natural repair ability, have wide clinical practice and market prospect.
Description of drawings
Fig. 1 is that SEM figure and the human umbilical cord mesenchymal stromal cell of the support of the microsphere porous property of PLGA of sintering preparation among the embodiment 2 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 RF among the figure of sequence number 1; Scale: sequence number 1 is respectively 100 μ m and 50 μ m with sequence number 2; Sequence number 3 and 4 figure are the real time imaging of human umbilical cord mesenchymal stromal cell when in support, cultivating for 2 weeks, 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 representes living cells, and the figure of sequence number 3 representes 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 to set forth the present invention, and protection scope of the present invention is not only to be confined to following examples.Content disclosed by the invention and scope that each parameter is got more than the those of ordinary skill foundation of said technical field all can realize the object of the invention.
Embodiment 1
1. make up homogenizing PLGA microsphere
The PLGA microsphere of preparation diameter 50 μ m.PLGA composition (mol ratio is the lactic acid of 1:1: glycolic, the molecular weight of polymer ~ 25,000) warp polymerization is at random processed PLGA; With dichloromethane DCM (DCM 30%W/V; Mass volume ratio) dissolving PLGA forms the PLGA lysate, and wherein the quality percentage composition of PLGA is 3% in the PLGA lysate, and this polymer solution is through ultrasonic sensor control waveform generator; Through the coaxial nozzle needle head of trumpet; Produce the homogeneous polymers drop, flow into and contain in the beaker of 0.5% (quality percentage composition) 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 subsequent use.
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) and obtain 0.2 μ g/mLBMP-2 stock solution at PBS (phosphate buffer).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 through ultrasonic sensor control waveform generator; Through the coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, flow into that to contain the quality percentage composition be in 0.5% the PVA beaker; Form TGF-β 3 and BMP-2/PLGA polymer drop; Earlier T GF-β 3 need stir 3-4 hour with BMP-2/PLGA polymer drop, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.Last TGF-β 3 and BMP-2/PLGA polymer microballoon lyophilizing 48 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is subsequent use.The TGF-β 3 and the BMP-2/PLGA microsphere of different-grain diameter collapse the flow speed control microspherulite diameter through microgranule, prepare in batches.
3. make up TGF-β 3/ BMP-2 gradient and discharge the PLGA microsphere support
With diameter be lyophilizing TGF-β 3 and BMP-2/PLGA microsphere and the blank PLGA microsphere of 50 μ m by mass and size (g/mL) than being 2.5% to be dispersed in ddH
2Among the O, wherein TGF-β 3 is that 1:1 is respectively charged into 20 mL syringes with the mass ratio of BMP-2/PLGA microsphere and blank PLGA microsphere.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, and (granule keeps>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, use infusion pump and vacuum syringe pump to make ddH at mould
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.
Embodiment 2
1. make up homogenizing PLGA microsphere
The PLGA microsphere of preparation diameter 100 μ m.PLGA composition (mol ratio is the lactic acid of 1:0.3: glycolic, the molecular weight of polymer ~ 25,000) warp polymerization is at random processed PLGA; With DCM (dichloromethane) (DCM 30%W/V; Mass volume ratio) dissolving PLGA forms the PLGA lysate, and wherein the quality percentage composition of PLGA is 1% in the PLGA lysate, and this polymer solution is through ultrasonic sensor control waveform generator; Through the coaxial nozzle needle head of trumpet; Produce the homogeneous polymers drop, flow into and contain in the beaker of 0.8% (quality percentage composition) 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 subsequent use.
2. make up TGF-β 3 and BMP-2/PLGA microsphere
Get BMP-2 adding bovine serum albumin (BSA) and obtain 0.3 μ g/mLBMP-2 stock solution at PBS (phosphate buffer).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 with the PLGA lysate respectively by volume; Ultrasonic agitation (50% vibration amplitude, 20 seconds) forms uniform PLGA protein emulsion on ice.This polymer solution is through ultrasonic sensor control waveform generator; Through the coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, flow into that to contain the quality percentage composition be in 0.3% the PVA beaker; Form TGF-β 3 and BMP-2/PLGA polymer drop; Earlier T GF-β 3 need stir 3-4 hour with BMP-2/PLGA polymer drop, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.Last TGF-β 3 and BMP-2/PLGA polymer microballoon lyophilizing 50 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is subsequent use.
TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support and prepares through 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 is dispersed in ddH with BMP-2/PLGA microsphere and blank PLGA microsphere
2O (2.5%W/V), wherein TGF-β 3 is that 1:1 is respectively charged into 20 mL syringes with the mass ratio of BMP-2/PLGA microsphere and blank PLGA microsphere.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, and (granule keeps>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, use infusion pump and vacuum syringe pump to make ddH at mould
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.PLGA composition (mol ratio is the lactic acid of 1:2: glycolic, the molecular weight of polymer ~ 25,000) warp polymerization is at random processed PLGA; With DCM (dichloromethane) (DCM 30%W/V; Mass volume ratio) dissolving PLGA forms the PLGA lysate, and wherein the quality percentage composition of PLGA is 5% in the PLGA lysate, and this polymer solution is through ultrasonic sensor control waveform generator; Through the coaxial nozzle needle head of trumpet; Produce the homogeneous polymers drop, flow into and contain in the beaker of 1.0% (quality percentage composition) 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 subsequent use.
2. make up TGF-β 3 and BMP-2/PLGA microsphere
Get BMP-2 adding bovine serum albumin (BSA) and obtain 0.4 μ g/mLBMP-2 stock solution at PBS (phosphate buffer).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 with the PLGA lysate respectively by volume; Ultrasonic agitation (50% vibration amplitude, 30 seconds) forms uniform PLGA protein emulsion on ice.This polymer solution is through ultrasonic sensor control waveform generator; Through the coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, flow into that to contain the quality percentage composition be in 0.8% the PVA beaker; Form TGF-β 3 and BMP-2/PLGA polymer drop; Earlier T GF-β 3 need stir 3-4 hour with BMP-2/PLGA polymer drop, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.Last TGF-β 3 and BMP-2/PLGA polymer microballoon lyophilizing 50 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is subsequent use.
TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support and prepares through 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 is dispersed in ddH with BMP-2/PLGA microsphere and blank PLGA microsphere
2O (2.5%W/V), wherein TGF-β 3 is 1:1 with the mass ratio of BMP-2/PLGA microsphere and blank PLGA microsphere, is respectively charged into the 20mL love dart.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, and (granule keeps>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, use infusion pump and vacuum syringe pump to make ddH at mould
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.PLGA composition (mol ratio is the lactic acid of 1:3: glycolic, the molecular weight of polymer ~ 25,000) warp polymerization is at random processed PLGA; With DCM (dichloromethane) (DCM 30%W/V; Mass volume ratio) dissolving PLGA forms the PLGA lysate, and wherein the quality percentage composition of PLGA is 2% in the PLGA lysate, and this polymer solution is through ultrasonic sensor control waveform generator; Through the coaxial nozzle needle head of trumpet; Produce the homogeneous polymers drop, flow into and contain in the beaker of 0.3% (quality percentage composition) 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 subsequent use.
2. make up TGF-β 3 and BMP-2/PLGA microsphere
Get BMP-2 adding bovine serum albumin (BSA) and obtain 0.5 μ g/mLBMP-2 stock solution at PBS (phosphate buffer).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 with the PLGA lysate respectively by volume; Ultrasonic agitation (60% vibration amplitude, 10 seconds) forms uniform PLGA protein emulsion on ice.This polymer solution is through ultrasonic sensor control waveform generator; Through the coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, flow into that to contain the quality percentage composition be in 1.0% the PVA beaker; Form TGF-β 3 and BMP-2/PLGA polymer drop; Earlier T GF-β 3 need stir 3-4 hour with BMP-2/PLGA polymer drop, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.Last TGF-β 3 and BMP-2/PLGA polymer microballoon lyophilizing 47 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is subsequent use.
TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support and prepares through 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 is dispersed in ddH with BMP-2/PLGA microsphere and blank PLGA microsphere
2O (2.5% W/ V), wherein TGF-β 3 is 1:1 with the mass ratio of BMP-2/PLGA microsphere and blank PLGA microsphere, is respectively charged into 20 mL love darts.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, and (granule keeps>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, use infusion pump and vacuum syringe pump to make ddH at mould
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.PLGA composition (mol ratio is the lactic acid of 1:0.5: glycolic, the molecular weight of polymer ~ 25,000) warp polymerization is at random processed PLGA; With DCM (dichloromethane) (DCM 30%W/V; Mass volume ratio) dissolving PLGA forms the PLGA lysate, and wherein the quality percentage composition of PLGA is 2.5% in the PLGA lysate, and this polymer solution is through ultrasonic sensor control waveform generator; Through the coaxial nozzle needle head of trumpet; Produce the homogeneous polymers drop, flow into and contain in the beaker of 0.6% (quality percentage composition) 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 subsequent use.The homogenizing PLGA microsphere of different-grain diameter collapses the flow speed control microspherulite diameter through microgranule, prepares in batches.
2. make up TGF-β 3 and BMP-2/PLGA microsphere
Get BMP-2 adding bovine serum albumin (BSA) and obtain 0.35 μ g/mLBMP-2 stock solution at PBS (phosphate buffer).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 with the PLGA lysate respectively by volume; Ultrasonic agitation (50% vibration amplitude, 30 seconds) forms uniform PLGA protein emulsion on ice.This polymer solution is through ultrasonic sensor control waveform generator; Through the coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, flow into that to contain the quality percentage composition be in 0.5% the PVA beaker; Form TGF-β 3 and BMP-2/PLGA polymer drop; Earlier T GF-β 3 need stir 3-4 hour with BMP-2/PLGA polymer drop, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.Last TGF-β 3 and BMP-2/PLGA polymer microballoon lyophilizing 49 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is subsequent use.
TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support and prepares through 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 is dispersed in ddH with BMP-2/PLGA microsphere and blank PLGA microsphere
2O (2.5%W/V, the mass volume ratio of microsphere and water), wherein TGF-β 3 is 1:1 with the mass ratio of BMP-2/PLGA microsphere and blank PLGA microsphere, is respectively charged into 20 mL love darts.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, and (granule keeps>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, use infusion pump and vacuum syringe pump to make ddH at mould
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.PLGA composition (mol ratio is the lactic acid of 1:1: glycolic, the molecular weight of polymer ~ 25,000) warp polymerization is at random processed PLGA; With DCM (dichloromethane) (DCM 30%W/V; Mass volume ratio) dissolving PLGA forms the PLGA lysate, and wherein the quality percentage composition of PLGA is 5% in the PLGA lysate, and this polymer solution is through ultrasonic sensor control waveform generator; Through the coaxial nozzle needle head of trumpet; Produce the homogeneous polymers drop, flow into and contain in the beaker of 0.5% (quality percentage composition) 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 subsequent use.The homogenizing PLGA microsphere of different-grain diameter collapses the flow speed control microspherulite diameter through microgranule, prepares in batches.
2. make up TGF-β 3 and BMP-2/PLGA microsphere
Get BMP-2 adding bovine serum albumin (BSA) and obtain 0.1 μ g/mLBMP-2 stock solution at PBS (phosphate buffer).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 with the PLGA lysate respectively by volume; Ultrasonic agitation (50% vibration amplitude, 30 seconds) forms uniform PLGA protein emulsion on ice.This polymer solution is through ultrasonic sensor control waveform generator; Through the coaxial nozzle needle head of trumpet, produce the homogeneous polymers drop, flow into that to contain the quality percentage composition be in 0.5% the PVA beaker; Form TGF-β 3 and BMP-2/PLGA polymer drop; Earlier T GF-β 3 need stir 3-4 hour with BMP-2/PLGA polymer drop, formed microsphere with after-hardening, filter the back with distilled water (~ 1L) to remove residual PVA.Last TGF-β 3 and BMP-2/PLGA polymer microballoon lyophilizing 48 hours ,-20 ℃ of storages are measured microsphere diameter and internal structure, form is subsequent use.
TGF-β 3/ BMP-2 gradient discharges the PLGA microsphere support and prepares through 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 is dispersed in ddH with BMP-2/PLGA microsphere and blank PLGA microsphere
2O (2.5%W/V, the mass volume ratio of microsphere and water), wherein TGF-β 3 is 1:1 with the mass ratio of BMP-2/PLGA microsphere and blank PLGA microsphere, is respectively charged into 20 mL love darts.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, and (granule keeps>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, use infusion pump and vacuum syringe pump to make ddH at mould
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.
The TGF-β 3/ BMP-2 gradient that below makes up for the foregoing description 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, Pharmacia company, the U.S.) on the Percoll separating medium; 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 subsequent use to get the 4th generation hBMSCs.
Table 1 experiment divides into groups to amount to 10 groups
| Factor | Levels |
| Microsphere diameter | 50,100,150,200,250,300 μ m |
| Somatomedin | TGF-β 3, 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 subsequent use 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.
The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is 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: at first prepare PLGA homogenizing microsphere and TGF-β 3 and BMP-2/PLGA microsphere through following method; Lyophilizing is subsequent use respectively, and freeze dried above-mentioned microsphere is scattered in ddH
2Process suspension among the O, then this suspension is injected the mould that the bottom is provided with defecator, adopt ddH
2O filters and adjustment 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 promptly 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 through following method: be 1:0.3-3 in molar ratio; Extracting lactic acid and hydroxyacetic acid polymerization at random generate PLGA, PLGA are dissolved in form the PLGA lysate among the dichloromethane DCM, and 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, and 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 prepares through following method with the BMP-2/PLGA microsphere: TGF-β 3 is dissolved in processes TGF-β 3 stock solutions among the bovine serum albumin BSA; BMP-2 is dissolved in processes BMP-2 stock solution among the bovine serum albumin BSA, PLGA is dissolved in forms the 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; Form TGF-β 3/BMP-2-PLGA microsphere after filtration, 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, and the quality percentage composition of PLGA is 1-5% in the lysate of PLGA, and 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, 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 a 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 method for preparing of microsphere support, it is characterized in that comprising following steps:
(1) preparation PLGA homogenizing microsphere, lyophilizing is subsequent use;
(2) preparation TGF-β 3 and BMP-2/PLGA microsphere, lyophilizing is subsequent use;
(3) freeze dried TGF-β 3 is scattered in ddH with BMP-2/PLGA microsphere and PLGA homogenizing microsphere
2Process suspension among the O, then this suspension is injected the mould that the bottom is provided with defecator, adopt ddH
2O filters and adjustment 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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