CN108159490A

CN108159490A - It is a kind of to promote the bone tissue engineering scaffold that blood vessel quickly generates

Info

Publication number: CN108159490A
Application number: CN201810041798.2A
Authority: CN
Inventors: 白燕; 丁庆
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2018-01-16
Filing date: 2018-01-16
Publication date: 2018-06-15

Abstract

The present invention is a kind of compound rest that bone tissue engineer medium vessels is promoted to quickly generate and its preparation.In the present invention, PLLA (l-lactic acid) microballoon of PDGF (platelet derived growth factor) is loaded with using Solution-enhanced dispersion by supercritical preparation, recycle supercritical carbon dioxide foaming technology that microballoon, VEGF (vascular endothelial growth factor) and FGF 2 (basic fibroblast growth factor) three are loaded into jointly in PLGA (polylactic glycolic) porous support, finally plant vascular endothelial cell on stent, structure can coordinated regulation angiogenesis compound rest.In the present invention, the vascular endothelial cell of plantation can be divided into new blood vessel, its extracellular matrix secreted can improve the adherency of relevant cell, and time release of three kinds of blood vessel inducible factors can induce the growth of blood vessel, and three's synergistic effect can promote quickly generating for Bone Defect Repari process medium vessels.Preparation manipulation of the present invention is simple, and obtained compound rest has good biocompatibility and functionality, has application prospect in organizational project dummy.

Description

It is a kind of to promote the bone tissue engineering scaffold that blood vessel quickly generates

Technical field

The present invention is a kind of compound rest that bone tissue engineer medium vessels is promoted to quickly generate and its preparation, belongs to drug Dosage form change and preparation method thereof technical field.Specifically, it is to be combined microballoon and stent using supercritical fluid technique To prepare the new method of intravascular tissue engineering stent, more particularly to Solution-enhanced dispersion by supercritical and supercritical foaming Technology is to tri- kinds of PDGF, FGF-2 and VEGF in the green controllable preparation process and compound rest of drug bearing microsphere and compound rest Time controlled release of blood vessel inducible factor.

Background technology

Blood vessel is not only the transport channel of oxygen, various nutritional ingredients and metabolic waste, and is to participate in tissue repair Cell signaling molecule by important channel, on the whole maintain bone tissue reparation metabolism microenvironment.Therefore, it is rapid effective Reconstructing blood vessel is that the premise of bone tissue regeneration and the key of guarantee and long-term restriction tissue engineered bone development and clinical practice are asked Topic.During angiogenesis, various growth factors are most important to the regulating and controlling effect of local organization, cell, apply quilt It is considered to solve one of most directly effective approach of engineering blood vesselization.The targeting cell of various blood vessel inducible factors and induction Mechanism is different, they are respectively acting on the different times of angiogenesis, and also there is mutual collaboration tune between the various factors The effect of control.Wherein, the effect of tri- kinds of growth factors of VEGF, FGF and PDGF is most important.VEGF is known induction of vascular life Into most significant growth factor is acted on, the proliferation of endothelial cell is promoted by the specific binding with endothelial cell membrane receptor, is divided Change, migration, additionally it is possible to enhance vasopermeability, inhibit proliferation and the migration of vascular smooth muscle cells, promote plasma substance to flat The sub- endodermis aggregation that sliding myocyte is formed.FGF-2 can promote proliferation and the differentiation of various kinds of cell, while adjust cell generation It thanks, the secretion including activator of plasminogen and clostridiopetidase A adjusts the expression and synthesis of other growth factors.PDGF be it is a kind of by The glycoprotein that blood platelet generates, as a kind of mitogen and chemotactic factor (CF), can promote fibroblast, smooth muscle thin simultaneously Born of the same parents and the division and growth of endothelial cell.Although these growth factors all have been confirmed the inducing action of angiogenesis, But single growth factor can not achieve regeneration truly, because tissue repair regeneration is a multiple-factor association With regulation and control as a result, how to realize that a variety of growth factors are crucial to the coordinated regulation of angiogenesis.

Growth factor to the coordinated regulation of angiogenesis and its type, combine, dosage, action time and mode etc. it is a variety of because It is known as pass.The early stage work study of this seminar VEGF, FGF-2 and PDGF are to human umbilical vein endothelial cell (HUVEC) Between blood vessel differentiation and the dose dependent of chick chorioallantoic membrane (CAM) medium vessels generation, time dependence and each factor Synergistic effect.The results show that VEGF and FGF-2 is to the early period that the best induction time of angiogenesis is angiogenesis, i.e., preceding 7 My god；And PDGF was to the middle and later periods that the best induction time of angiogenesis is angiogenesis, i.e., 7-21 days.Three kinds of agents applications There is significant co-induction effect to angiogenesis and skeletonization.However, directly use growth factor there are half-life short, easily Inactivation, deficient in stability and the problems such as tissue selectivity, and can not according to tissue growth different phase need releasing by parts, Coordinated regulation.There are a variety of growth factors time the to release therefore, it is necessary to the inducing properties according to the different factors and mechanism structure are a kind of Put the carrier system of characteristic, make its simulated blood vessel formed with regenerated physiology course, timely and appropriately discharge different growths because Son arrives the tissue and cell needed.But not yet reported examples of such carriers system.This research is assisted based on different growth factors With regulation and control revascularization mechanism, the characteristics of with reference to microballoon and stent slow-released system respectively, prepare a kind of release initial stage with VEGF and Based on FGF-2, organizational project of the middle and later periods based on PDGF makes it effectively facilitate bone tissue engineer medium vessels and quickly generates.

On the construction method of growth factor carrier system, traditional method for example emulsion process, spray drying process, fusion method, Salting out method etc., the problems such as being often accompanied by high temperature and pressure, severe reaction conditions, stability and bioactivity to protein medicaments generate Large effect.Therefore, in order to maximize holding protein active, using a kind of mild process conditions method come prepare carry growth because The polymer support of son is necessary.Supercritical fluid technique meets this requirement intelligently.Supercritical carbon dioxide fluid Technology is carried because of the features such as its is nontoxic and pollution-free, cost is relatively low, reaction condition is mild, product organic solvent-free remains, environmentally protective The more clean effective technical advantage of opposite conventional method has been supplied, this method can maximumlly keep growth factor activity, More aobvious unique advantage in terms of the preparation of protein medicaments carrier.But at present, by supercritical fluid technique for complex microsphere and The preparation of a variety of growth factor carrier systems of stent not yet appears in the newspapers.

The purpose of invention

According to the induction mechanism and action characteristic of different blood vessel inducible factor, using supercritical fluid technique by microballoon and branch Frame carries out a kind of compound polymer carrier system for preparing simulation physiology and repairing the different growth factors of situation pattern release, quickly has Effect ground promotes the generation of tissue engineered bone medium vessels.

The basic conception of invention

Growth factor slow-release system cannot only limit to the single growth factor of release, and should be in a kind of similar physiology reparation A variety of growth factors are discharged to the targeting cell needed with best ratio under situation pattern.The present invention is cooperateed with according to growth factor The best induction pattern of modulating vascular generation, polylactic acid is loaded into using supercritical carbon dioxide Forced Dispersion technology by PDGF (PLLA) in microballoon, then using supercritical carbon dioxide foaming technology by PDGF-PLLA microballoons, VEGF and FGF-2 freeze-dried powders carry Enter and more growth factor slow-release systems are built in compound rest, make the secondary release stage by stage of its release characteristics and dosage according to design A variety of growth factors, to meet the time under revascularization physiological status and growth factor needs spatially, so as to have rapidly Realize the vascularization of tissue engineered bone in effect ground.

Invention content

It is an object of the invention to the induction mechanism according to different factor pair angiogenesis and synergistic effect to prepare a kind of mould Intend physiology and repair the compound rest that situation mould discharges a variety of growth factors.It will using supercritical carbon dioxide Forced Dispersion technology PDGF be loaded into polylactic acid (PLLA) microballoon in, then using supercritical carbon dioxide foaming technology by PDGF-PLLA microballoons, VEGF and FGF-2 freeze-dried powders are loaded into compound rest and build more growth factor slow-release systems, make its release characteristics and dosage according to design The a variety of growth factors of secondary release stage by stage.It is tissue engineered bone so as to quickly and effectively realize the vascularization of tissue engineered bone Further application provide technical guarantee.

Technique used by a kind of compound rest that bone tissue engineer medium vessels is promoted to quickly generate prepared by the present invention Step is as follows：(1) preparation of PDGF-PLLA microballoons：PDGF is dissolved in dimethyl sulfoxide, PLLA is dissolved in dichloromethane.When two kinds After solution is all completely dissolved, the two is uniformly mixed.Mixed solution is pumped into high pressure by high performance liquid chromatography (HPLC) In deposition reaction kettle, Solution-enhanced dispersion by supercritical (SEDS) process is undergone.The microballoon of Precipitation stores up after collecting It is stored in -20 DEG C for use.

(2) preparation of compound rest：PLGA and hydroxyapatite (HA) are ground according to a certain percentage, added in a certain amount of (FGF-2 and VEGF freeze-dried powders are first with 10 000r/min or 12 for PDGF-PLLA drug bearing microspheres and FGF-2 and VEGF freeze-dried powders 000r/min centrifuges 30s, is resuspended in pH 7.6,5mmol/L Tris to 0.1g/L, add in PLGA, HA for mixing in advance and Gently it is lyophilized after mixing in PDGF-PLLA microsphere powders), mixed-powder is put into room temperature in the cylindrical die of a diameter of 5mm Under suppressed to obtain sequin, take out.It is subsequently placed in polytetrafluoroethylene (PTFE) cylinder mold and is put into mold overcritical CO₂In stainless steel cauldron, using supercritical CO₂Foaming technique prepares porous compound support frame.(2) kind of vascular endothelial cell It plants：The compound rest of a diameter of 5mm is positioned in 24 orifice plates, ultraviolet light irradiation 0.5h.Again with 75% alcohol disinfecting, PBS leachings Bubble cleaning 3 times.Sterile compound rest is placed in DMEM culture solutions and is incubated overnight.4th generation vascular endothelial cell is by training After supporting digestion, it is 1 × 10 to be prepared into density⁵The cell suspension of/ml is planted respectively on the timbering material being ready for, 37 DEG C, 5%CO₂Continue culture in cell incubator for 24 hours.

Original and significant technological progress of the invention is：

Based on a kind of preparation for the compound rest that bone tissue engineer medium vessels is promoted to quickly generate, on the one hand, utilize super Critical fluids Forced Dispersion solution technique prepares the PLLA microballoons for carrying PDGF, and will be micro- using supercritical carbon dioxide foaming technology Ball and VEGF, FGF-2 are loaded into stent jointly prepares compound rest.The performance of regulation and control compound rest is not only able to, is also effectively protected The bioactivity of growth factor is hindered, and available for the preparation of other protide macromolecule medicament carriers.On the other hand, it is of the invention The compound rest of preparation combines the advantage of microballoon and stent, and simulation physiology is repaired pattern release PDGF, FGF-2 and VEGF, filled The synergistic effects of three kinds of growth factors is waved in distribution, meets the needs of body in angiogenesis to different growth factors, and then Fast and effeciently promote the generation of tissue engineered bone medium vessels.Finally, the vascular endothelial cell planted on stent can be divided into newly Blood vessel, the extracellular matrix of secretion can improve the adherency of relevant cell, and the formation for new blood vessel provides cell base.

The purposes of the present invention：The obtained compound rest of the method for the present invention protide macromolecular drug delivery vector, Regenerative medicine field has great impetus and application prospect.

Description of the drawings

The preparation process schematic diagram of Fig. 1 compound rests

Fig. 2 Solution-enhanced dispersion by supercritical prepares PDGF-PLLA microballoons

Fig. 3 PDGF-PLLA drug bearing microsphere patterns

Fig. 4 compound rest patterns

PDGF, VEGF and FGF-2 release rule in Fig. 5 compound rests

Effect of Fig. 6 compound rests to chick embryo allantois chorion (CAM) angiogenesis

Specific embodiment

Below in conjunction with the accompanying drawings and following embodiment is described in further details the present invention, but can not limit the present invention's Content.Under the concept thereof of the present invention, the simple modifications of method of modifying of the present invention are belonged to claimed model It encloses.

The preparation of one PDGF-PLLA drug bearing microspheres of example

0.2mg PDGF are dissolved in 2.5mL dimethyl sulfoxides, 200mg molecular weight is that the PLLA of 10ku is dissolved in 40mL dichloromethanes In alkane.After two kinds of solution are all completely dissolved, the two is uniformly mixed for use.By the CO in steel cylinder₂After refrigeration system liquefies, Pressurizeed by high-pressure plunger pump, then by the water bath with thermostatic control heating in pipeline after, pass through on the outside of coaxial two stream nozzle of autoclave Channel is pumped in the autoclave that volume is 500mL.After pressure reaches 12Mpa in kettle, CO is maintained₂Rate is pumped into, unlatching is put Air valve is deflated with the flow velocity of 300ml/min, maintains pressure in kettle constant.Adjust drying box and pipeline water-bath temperature outside autoclave Degree, to keep temperature in the kettle constant at 33 DEG C.Then it is mixed testing liquid is logical by high performance liquid chromatography (HPLC) pump It crosses nozzle inner channel to be pumped into autoclave, testing liquid flow velocity is 0.5ml/min.Touch SCCO₂Afterwards, solvent is by SCCO₂Extraction It takes so that testing liquid becomes the supersaturated solution of solute, and the form crystallization of microballoon is precipitated solute from solution.Terminate pump sample Afterwards, it maintains pressure and temperature constant, continues to be passed through CO₂30 minutes are eluted to remove residual organic solvent.After terminating cleaning, slowly Release when pressure is reduced to normal pressure in kettle, opens reaction kettle and collects sample.It is stored in -20 DEG C for use.Scanning electron microscopic observation microballoon Pattern, as shown in Figure 1.

The preparation of two compound rest of example

By PLGA (PLA:PLG=50:50, MW=ku) with HA powder with 1:4 ratio is ground, milling time 8 Hour.Add in 20% PDGF-PLLA drug bearing microspheres and 20 μ g FGF-2 and VEGF freeze-dried powders.FGF-2 and VEGF freeze-dried powders First with 10 000r/min or 12 000r/min, 30s is centrifuged, is resuspended in pH 7.6,5mmol/L Tris to 0.1g/L, added in It is gently lyophilized after mixing in PLGA, HA and PDGF-PLLA microsphere powder mixed in advance.Mixed-powder is put into a diameter of It is suppressed to obtain sequin at room temperature in the cylindrical die of 5mm, be taken out.It is subsequently placed in polytetrafluoroethylene (PTFE) cylinder mold And mold is put into supercritical CO₂In stainless steel cauldron.It will be condensed to less than 0 DEG C of CO with liquid pump after reaction kettle sealing₂Press-in In reaction kettle, reaction pressure starts to warm up when being 8MPa, and reaction temperature is 35 DEG C, passes through the thermocouple and blow valve built in instrument The pressure and temperature in kettle is adjusted, temperature change is kept to be no more than ± 0.5 DEG C, pressure change is ± 0.1Mpa.After reacting 8h, with Certain rate is deflated, decompression rate 0.1MPa/s.Reaction kettle is opened, takes out sample, is stored in -20 DEG C for use.Scanning electricity Sem observation microballoon pattern, as shown in Figure 2.Detect the accumulative release rate of PDGF, VEGF and FGF-2 in compound rest, such as attached drawing Shown in 3.

The induction that example triplex stent generates chick embryo allantois chorion medium vessels

Surface cleaning is selected, the white skin hatching egg of eggshell homogeneous, per egg 50-65g, shape specification, gas chamber, stomata are uniform.With Warm water cleans 2 times, is then impregnated 3 minutes with bromogeramine washing lotion.Chicken embryo is positioned in incubator and is incubated, temperature 37.8 ± 0.5 DEG C, humidity 40-60%.During incubation will ceaselessly turning egg(s), can prevent embryo from sticking together, promote amnion movement.Chicken Embryo end is upward, in 45.It tilts, daily at least turning egg(s) 2-4 times.Embryonic development situation is looked into inspection egg lamp inspection daily, identifies embryo The symbolic characteristic of development eliminates stillborn foetus at any time.Be incubated be within the 2-6 days chick embryo development critical period, hereafter embryo itself has been There is body temperature, and with certain regulating power.In order to improve the survival rate of chicken embryo, start within the 7th day dosing being incubated and be conducive to pair Pharmaceutically-active vascular effect to be measured is observed.The characteristics of chicken embryo medium vessels are grown is to be the 3-5 days, 7-10 days of incubation Angiogenic growth peak period, angiogenic growth is most active, convenient for the observation of result；The gradual atrophy of CAM blood vessels after 12 days；5-6 days left The right side, chorion are merged with chorioallantoic membrane, therefore choose 7-12 days as medicine irritation Best Times.Therefore, it selects the 7th day to the 12nd It carries out investigation of the growth factor to angiogenic effect.It with 75% alcohol wipe chorion, is used after being dried up in super-clean bench within 7th day Aseptic nipper and sharp mouth cone carefully drill out a duck eye in the flat blunt end of egg, are carefully covered with sterile parafilm with anti-drying, The flat blunt end of chicken embryo is upward in 37 DEG C, 55% relative air humidity and 3%CO₂Under conditions of continue to be incubated, but no longer stir.It opens Second day after window, compound rest is added according to experiment packet, with the chicken embryo developed naturally as a control group.Remove chicken within 13rd day Embryo is removed CAM and is detached with egg skin, is observed with disecting microscope and counts angiogenesis situation, as shown in Figure 4.

Claims

1. a kind of preparation method for the compound rest that bone tissue engineer medium vessels is promoted to quickly generate, which is characterized in that described Method includes the following steps：

(1) preparation of PDGF-PLLA microballoons：PDGF is dissolved in dimethyl sulfoxide, PLLA is dissolved in dichloromethane.When two kinds of solution all After being completely dissolved, the two is uniformly mixed.Mixed solution is pumped into high pressure deposition instead by high performance liquid chromatography (HPLC) It answers in kettle, undergoes Solution-enhanced dispersion by supercritical (SEDS) process.The microballoon of Precipitation is stored in -20 after collecting It is DEG C for use.

(2) preparation of compound rest：Hydroxyapatite (HA) and PLGA are pressed 1:4-1:2 grindings, add in a certain amount of PDGF- (FGF-2 and VEGF freeze-dried powders are first with 10 000r/min or 12 000r/ for PLLA drug bearing microspheres and FGF-2 and VEGF freeze-dried powders Min centrifuges 30s, is resuspended in pH 7.6,5mmol/L Tris to 0.1g/L, adds in PLGA, HA and the PDGF- mixed in advance Gently be lyophilized after mixing in PLLA microsphere powders), by mixed-powder be put into the cylindrical die of a diameter of 1-8mm at room temperature into Row compacting obtains sequin, takes out.It is subsequently placed in polytetrafluoroethylene (PTFE) cylinder mold and mold is put into supercritical CO₂It is stainless In steel reaction kettle, using supercritical CO₂Foaming technique prepares porous compound support frame.

(3) plantation of vascular endothelial cell：Compound rest is positioned in 24 orifice plates, ultraviolet light irradiation 0.5h.Again with 75% wine Essence disinfection, PBS soaking and washings 3 times.Sterile compound rest is placed in DMEM culture solutions and is incubated overnight.In 4th generation, is intravascular For chrotoplast after culture digests, it is 1 × 10 to be prepared into density⁵The cell suspension of/ml is planted respectively in the stent being ready for On material, 37 DEG C, 5%CO₂Continue culture in cell incubator for 24 hours.

2. a kind of preparation side of compound rest that bone tissue engineer medium vessels is promoted to quickly generate according to claim 1 Method, which is characterized in that in step 1, when preparing PDGF-PLLA microballoons using SEDS, will be tested respectively by double-channel spray nozzle molten Liquid and supercritical CO₂(SCCO₂) spray into autoclave.When solution touches SCCO₂Afterwards, solvent is by SCCO₂Extraction so that Testing liquid becomes the supersaturated solution of solute, and the form crystallization of microballoon is precipitated solute from solution.After terminating pump sample, maintain Pressure and temperature is constant, continues to be passed through CO₂30-60 minutes are eluted to remove residual organic solvent.

3. a kind of preparation side of compound rest that bone tissue engineer medium vessels is promoted to quickly generate according to claim 1 Method, which is characterized in that in step 1, use SEDS prepare the reaction conditions of PDGF-PLLA microballoons for：31-35 DEG C of temperature, pressure 8-15Mpa, SCCO₂Flow velocity 200-400ml/min, testing liquid flow velocity are 0.1-0.5ml/min.

4. a kind of preparation side of compound rest that bone tissue engineer medium vessels is promoted to quickly generate according to claim 1 Method, which is characterized in that in step 1, when preparing PDGF-PLLA microballoons using SEDS：The dosage of PDGF be 0.1-0.2mg, diformazan The dosage of base sulfoxide is 1-2.5mL.The molecular weight of PLLA is 1-10ku, dosage 100-200mg, and the dosage of dichloromethane is 10-40mL。

5. a kind of preparation side of compound rest that bone tissue engineer medium vessels is promoted to quickly generate according to claim 1 Method, which is characterized in that in step 2, using supercritical CO₂The reaction condition that foaming technique prepares porous compound support frame is：Sealing It will be condensed to less than 0 DEG C of CO with liquid pump afterwards₂It is pressed into reaction kettle, reaction pressure starts to warm up when being 8-10MPa, reaction temperature It is 30-37 DEG C, by the pressure and temperature in the thermocouple built in instrument and emptying valve regulation kettle, temperature change is kept to be no more than ± 0.5 DEG C, pressure change is ± 0.1Mpa.After reacting 8h, deflated with certain rate, decompression rate 0.1-0.5MPa/s.

6. a kind of preparation side of compound rest that bone tissue engineer medium vessels is promoted to quickly generate according to claim 1 Method, which is characterized in that in step 2, using supercritical CO₂When foaming technique prepares porous compound support frame：The molecular weight of PLGA is 1-100ku, block ratio 50:50-75:50, HA dosage is 2-10%, and the dosage of PDGF-PLLA microballoons is 5-40%, FGF- The dosage of 2 and VEGF freeze-dried powders is 10-50 μ g.

7. a kind of preparation side of compound rest that bone tissue engineer medium vessels is promoted to quickly generate according to claim 1 Method, which is characterized in that release the 1st to 7 day, the accumulative release rate of VEGF, FGF-2 and PDGF are respectively 71.10,69.76 and 43.17%.It discharges the 7th to 21 day, the accumulative release rate of VEGF, FGF-2 and PDGF are respectively 11.14,10.91 and 28.49%. Growth factor release initial stage is with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) Main, the middle and later periods based on platelet derived growth factor (PDGF), disclosure satisfy that angiogenic growth different times to growth factor It needs, realizes that growth factor acts on the co-induction of revascularization.