CN103919733A - Microsphere in core-shell structure wrapping double growth factors and use of microsphere - Google Patents
Microsphere in core-shell structure wrapping double growth factors and use of microsphere Download PDFInfo
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Abstract
The invention discloses a microsphere in a core-shell structure wrapping double growth factors and use of the microsphere. The microsphere comprises a core-shell spherical structure, wherein the core structure is made from polylactic acid, the viscosity of which is 0.5-2.0dL/g; the shell structure is made from a composite material consisting of polylactic acid and polyglycolic acid, the weight ratio of which is (75-50):(25-50). The core and shell respectively wrap a growth factor which is BMP-7 (Bone Morphogenetic Protein) or PDGF (Platelet-Derived Growth Factor)-BB. The invention further provides an application of the microsphere in the core-shell structure wrapping double growth factors in preparing a mesenchymal stem cell osteogenic differentiation medicine for inducing cell proliferation and low cell density culture. The microsphere in the core-shell structure wraps two growth factors at the same and releases the two growth factors successively, so that the two growth factors are respectively released in the most proper time with a good effect.
Description
Technical field
The present invention relates to functional living being material technology field, be specifically related to a kind of nucleocapsid structure microsphere that wraps up two somatomedin and uses thereof.
Background technology
Union of fracture or reparation are one and are subject to multiple somatomedin (Growth factor, GF) process of comprehensive regulation, wherein, FGF-2m (Fibroblast growth factor, FGF-2) with platelet derived growth factor (PLLAtelet-derived growth factor, etc. PDGF-BB) regulate cell enlargement and division, it is osteoblast precursor that bone morphogenetic protein (Bone morphogenetic protein, BMPs) etc. can be induced a Derived from Mesenchymal Stem Cells.There are some researches show, in bone defect repair process, two or more somatomedin couplings are better than the effect of alone a kind of somatomedin.But in the damaged natural repair process of bone, FGF-2m (FGF-2) and platelet derived growth factor (PDGF-BB) etc. are mainly being repaired early stage appearance, after bone break up and the osseous maturation stage does not express; Bone morphogenetic protein (BMPs) is mainly expressed in the bone formation stage.Rat bone extirpation experiment shows, with external source FGF-2m (FGF-2) short time (as 1 week) interaction energy promotion angiogenesis, increase callus volume, stimulate and produce mineralization, but long duration of action can postpone cell Osteoblast Differentiation and ripe mineralising.Possible reason is, FGF-2m (FGF-2) plays an important role to short cell proliferation, but this ability stops along with the formation of chondrocyte, needs other somatomedin to be used for continuing Osteoblast Differentiation.These researchs show, design controlled-release material carries growth factor [such as platelet derived growth factor (PDGF-BB)] and bone differentiation factor [such as bone morphogenesis protein-7 (BMP-7)] and imitates bone nature repair process to carry out sequential release experiment in body be successfully to break through necessity and the crucial method of osteanagenesis strategy.The simple immixture of a kind of somatomedin or several somatomedin is only considered in existing most research, has limited development.Current technology can not well be controlled two kinds of materials and accurately discharge.Therefore, designing the equipped system that two kinds of factors of new control discharge is necessary.
Summary of the invention
For the deficiencies in the prior art, the object of the present invention is to provide a kind of nucleocapsid structure microsphere that wraps up two somatomedin, this nucleocapsid structure microsphere can wrap up two kinds of somatomedin (such as platelet derived growth factor (PDGF-BB) and bone morphogenesis protein-7 (BMP-7)) simultaneously, and successively discharge this two kinds of somatomedin, the rhythm occurring with two kinds of somatomedin in fracture normal healing process agrees with, and this nucleocapsid structure microsphere can efficiently be induced mescenchymal stem cell Osteoblast Differentiation between the bone marrow that low cell density cultivates.The nucleocapsid structure microsphere that another object of the present invention is to provide the two somatomedin of described parcel is being prepared inducing cell propagation and is being induced the application aspect mescenchymal stem cell Osteoblast Differentiation medicine between the bone marrow that low cell density cultivates.The application's core-shell is double-deck microsphere supported can be controlled multiple somatomedin control and discharge, and is conducive to study the coordinative role relation between somatomedin, for organizational project has been opened up a new direction.Adopt and strictly control in this way cell proliferation and Osteoblast Differentiation, be not only conducive to study the coordinative role of these two kinds of physiological activities, more clinical treatment large area bone is damaged provides new medicine.
To achieve these goals, the present invention has adopted following technical scheme:
A nucleocapsid structure microsphere that wraps up two somatomedin, comprises nucleocapsid spherical structure, and described core is polylactic acid, and polylactic acid viscosity is 0.5-2.0dL/g; Shell is the composite of polylactic acid and polyglycolic acid composition, and in described composite, the mass ratio of polylactic acid and polyglycolic acid is: 75-50:25-50; In described core and shell, be enclosed with respectively a kind of somatomedin, described somatomedin is BMP-7 or PDGF-BB.
Preferred version: the somatomedin of described core parcel is BMP-7, the somatomedin of described shell parcel is PDGF-BB.
Preferred version: the external diameter of described nucleocapsid structure microsphere is 18-22 micron, the mass ratio of described core and shell is: 1:3-4, the mass percent of somatomedin in core is 0.002%-0.005%, and the mass percent of somatomedin in shell is 0.0008%-0.00125%.
More preferably scheme: the core of described nucleocapsid structure microsphere and the mass ratio of shell are: 1:4, it is 0.003% that the mass percent of somatomedin in core and shell is respectively the mass percent of somatomedin in core, the mass percent of somatomedin in shell is 0.001%.。
Preferred version: described polylactic acid viscosity is 1.0dL/g.
Preferred version: the mass ratio of described polylactic acid and polyglycolic acid is: 50:50.
The nucleocapsid structure microsphere of the two somatomedin of described parcel preferably prepares by the following method:
(1) PDGF-BB is dissolved in to deionized water and makes the water I that concentration is 1 μ g/ml-100 μ g/ml, composite is dissolved in to dichloromethane and makes the organic facies I that quality percent by volume is 8%-12%, water I and organic facies I mix rear ultrasonic emulsification for 1:8-12 by volume, obtain emulsion I; By same method, BMP-7 is dissolved in to deionized water and makes the water II that concentration is 1 μ g/ml-100 μ g/ml, polylactic acid is dissolved in to ethyl acetate and prepares the organic facies II that quality percent by volume is 2%-5%, water II and organic facies II mix rear ultrasonic emulsification for 1:8-12 by volume, obtain emulsion II;
(2) adopt coaxial electrostatic spray technique, emulsion I and emulsion II are sprayed into respectively in the outer pin and interior pin of coaxial needles of electrostatic atomizer, in controlling, in pin, the flow of material is 1.0-1.2ml/h, the flow of controlling material in outer pin is 1.5-2.0ml/h, 10cm-12cm place below coaxial needles, the microsphere of preparing with the culture dish reception of filling dehydrated alcohol, then washing, centrifugal again, lyophilization.
In interior pin and outer pin, the flow of solution is very crucial, and in interior pin, the flow of material is 1.0-1.2ml/h, and in outer pin, the flow of material is that 1.5-2.0ml/h relatively optimizes, if the flow of outer pin is too little, can not form nucleocapsid structure.
Preferred version: in described water I, the concentration of PDGF-BB is 5 μ g/ml-20 μ g/ml, and in described organic facies I, the quality percent by volume of composite is 10%; In described water II, the concentration of BMP-7 is 5 μ g/ml-20 μ g/ml, and in described organic facies II, the quality percent by volume of composite is 5%.
The application also provides the nucleocapsid structure microsphere of the two somatomedin of described parcel in the application of preparing in inducing cell propagation medicine.
The application also provides nucleocapsid structure microsphere application in mescenchymal stem cell Osteoblast Differentiation medicine between the bone marrow of the low cell density cultivation of preparation induction of the two somatomedin of described parcel.
The application also provides the nucleocapsid structure microsphere of the two somatomedin of described parcel to treat the application in fracture and injury medicine in preparation.
The invention discloses a kind of method of utilizing double-deck core-shell particles simulating nature osteanagenesis process microenvironment, described method adopts coaxial electrostatic spray technique and coaxial electrostatic spraying apparatus (existing equipment), polylactic acid (PLLA)/composite (PLGA) core-shell particles of bone morphogenesis protein-7 (BMP-7) and platelet derived growth factor (PDGF-BB) is carried in preparation, is embedded in respectively in the core and shell of microsphere by bone morphogenesis protein-7 (BMP-7) and platelet derived growth factor (PDGF-BB).
Described coaxial electrostatic spraying apparatus, has adopted coaxial needles to be made up of 316L rustless steel.External capillary (outer capillary) external diameter is 0.72mm, and internal diameter is 0.50mm, and nucleation capillary tube (core capillary) external diameter is that 0.40m and internal diameter are 0.20mm.There are two syringe pumps to arrive the inside and outside capillary tube of coaxial needles with special speed transfer polymer solution, make the microsphere forming there is core-shell structure.Voltage generator is supplied high voltage by crocodile clip to nozzle.For stabilized nozzle electric field around, apply high voltage to nozzle plug ring (diameter 5cm) around.By increasing spray nozzle voltage (V
nozzle) and plug ring voltage (V
ring) form stable electrical potential difference, form stable spraying.
Compared with prior art, advantage of the present invention is:
The present invention sprays two kinds of different somatomedin to be encapsulated in double-deck shell-core structure microsphere by coaxial electrostatic, realizes the different parts that two kinds of somatomedin is encapsulated in to microsphere by a step, realizes the control of two kinds of somatomedin is discharged.Control and discharge two kinds of somatomedin, the better natural microenvironment of the osteanagenesis in analogue body, to promoting osteanagenesis to play maximum effect.Existing many osteanagenesis therapies can only discharge a kind of somatomedin or simple mixing discharges two kinds of somatomedin.Therefore, the present invention realizes two kinds of somatomedin of embedding at microsphere different parts by a step, different somatomedin are discharged with different release profiles at different time, mix delivery systme with existing monofactor delivery systme and simple two kinds of factors and compare, successively discharge the better effects if of the system promoting bone regeneration of two kinds of somatomedin.
Brief description of the drawings
Fig. 1 is the impacts of various somatomedin on mescenchymal stem cell (MSC) propagation
Fig. 2 is the impact that various somatomedin distribute on mescenchymal stem cell (MSC) growth cycle
Fig. 3 is the impacts of various somatomedin on mescenchymal stem cell (MSC) Osteoblast Differentiation alkaline phosphatase activities
Fig. 4 is the impacts of various somatomedin on mescenchymal stem cell (MSC) Osteoblast Differentiation calcium deposition
Fig. 5 is the impact of PLLA/PGA composition on BMP-7 release profiles in Shell Materials in core-shell particles system
Fig. 6 is the impact of PLLA viscosity on PDGF-BB release profiles in core-shell particles system center layer material
Fig. 7 is the impact of PLLA/PGA composition on PDGF-BB release profiles in Shell Materials in core-shell particles system
Fig. 8 is the impact of PLLA viscosity on BMP-7 release profiles in core-shell particles system center layer material
Fig. 9 is the PDGF-BB release profiles of different microsphere systems
Figure 10 is the BMP-7 release profiles of different microsphere systems
Figure 11 is the ALP intensity of mescenchymal stem cell (MSC) after processing with different microspheres
The calcium concentration of mescenchymal stem cell (MSC) after Figure 12 processes with different microspheres
Figure 13 is the preparation process schematic diagram of microsphere, and wherein 1 is high voltage power supply, the 2nd, and internal emulsification liquid pump, the 3rd, outer emulsion pump, the 4th, outer pin, the 5th, interior pin, the 6th, culture dish.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further explained and is illustrated
Embodiment 1:
A nucleocapsid structure microsphere that wraps up two somatomedin, comprises nucleocapsid spherical structure, and described core is polylactic acid, and polylactic acid viscosity is 0.5-2.0dL/g; Shell is the composite of polylactic acid and polyglycolic acid composition, and in described composite, the mass ratio of polylactic acid and polyglycolic acid is: 75-50:25-50; In described core and shell, be enclosed with respectively a kind of somatomedin, described somatomedin is BMP-7 or PDGF-BB.
Wrap up a concrete preparation process for the nucleocapsid structure microsphere of two somatomedin, as shown in figure 13:
(1) PDGF-BB be dissolved in to deionized water make the water of concentration 10 μ g/ml; Composite is dissolved in to dichloromethane and makes the organic facies that quality percent by volume (grams per milliliter) is 10%, water and organic facies by volume percentage ratio are ultrasonic emulsification after 1:10 mixes, and obtain emulsion I; By same method, BMP-7 is dissolved in to deionized water and makes the water of concentration 10 μ g/ml; Polylactic acid is dissolved in to ethyl acetate and prepares the organic facies that mass volume ratio percentage ratio (grams per milliliter) is 3%, water and organic facies are mixed rear ultrasonic emulsification for 1:10 by volume, obtain emulsion II; In described composite, the mass ratio of polylactic acid-polyglycolic acid is: 50:50;
(2) adopt coaxial electrostatic spray technique, emulsion I and emulsion II are sprayed into respectively in the outer pin and interior pin of coaxial needles of electrostatic atomizer, in controlling, in pin, the flow of material is 1.2ml/h, the flow of controlling material in outer pin is 1.5ml/h, 10cm place below coaxial needles, the microsphere of preparing with the culture dish reception of filling dehydrated alcohol, then washing, centrifugal again, lyophilization.
The coaxial needles of electrostatic atomizer is made up of 316L rustless steel, the external diameter of shell needle tubing processed is 0.72mm, internal diameter is 0.50mm, the external diameter of core needle tubing processed is that 0.40mm and internal diameter are 0.20mm, device sprays into coaxial needles by PDGF-BB/ nuclear material, BMP-7/ shell material by different pipelines respectively by two syringe pumps, 10cm place below coaxial needles, the microsphere of preparing with the culture dish reception of filling dehydrated alcohol, in case microsphere cohesion.The microsphere of collecting is centrifugal rear by washed with de-ionized water at a slow speed, lyophilizing after recentrifuge.
The external diameter of described nucleocapsid structure microsphere is 20 microns, and the mass ratio of described core and shell is: 1:4, and the mass percent of somatomedin in core is 0.0033%, the mass percent of somatomedin in shell is 0.001%.
Comparative example: fixing other factors is constant, and preparation method is with embodiment 1, and the position that only changes the somatomedin of parcel location or parcel compares experiment:
Both somatomedin are wrapped in common monolayer micro-sphere structure simultaneously, and the single shaft syringe needle preparation that monolayer microsphere is 0.50mm by internal diameter, hereinafter to be referred as U;
Somatomedin (PDGF-BB) is wrapped in the double-deck microsphere with core-shell structure simultaneously, has only wrapped up a kind of somatomedin hereinafter to be referred as PB();
Somatomedin (BMP-7) is wrapped in the double-deck microsphere with core-shell structure simultaneously, has only wrapped up a kind of somatomedin hereinafter to be referred as B();
Somatomedin (BMP-7) and somatomedin (PDGF-BB) are wrapped in the double-deck microsphere with core-shell structure simultaneously, are wrapped in double-layer structure hereinafter to be referred as P(two somatomedin simultaneously);
Somatomedin PDGF-BB is wrapped in the stratum nucleare material of core-shell structure, BMP-7 is wrapped in the Shell Materials of core-shell structure, hereinafter to be referred as S1 simultaneously;
Growth factor B MP-7 is wrapped in the stratum nucleare material of core-shell structure, PDGF-BB is wrapped in the Shell Materials of core-shell structure, hereinafter to be referred as S2 simultaneously.
Embodiment 2 applied researcies
Mescenchymal stem cell (MSC) proliferation assay between 2.1 cytokines are short
By the promoting growth of cell ability of the cell counting measuring exogenous growth factor, platelet derived growth factor (PDGF-BB) between the impact of mescenchymal stem cell (MSC) cell proliferation there is stage, as shown in Figure 1, when concentration is during lower than 20ng/mL, cell number and platelet derived growth factor (PDGF-BB) concentration is linear, but while strengthening concentration again, short ability of cell proliferation obviously weakens, and the short propagation amplitude of 20ng/mL platelet derived growth factor (PDGF-BB) is about 50%.Contrary, bone morphogenesis protein-7 (BMP-7) presents interim depressing proliferation effect, when low concentration (0.1-1.0ng/ml), promotion and the inhibitory action of bone morphogenesis protein-7 (BMP-7) on cell proliferation are all not obvious, when concentration high (10-200ng/ml), can suppress cell proliferation, and inhibition is relevant with dosage.
Many experiments result shows, when 20ng/mL(fixed value) while adding bone morphogenesis protein-7 (BMP-7) in platelet derived growth factor (PDGF-BB), the ability of finding bone morphogenesis protein-7 (BMP-7) inhibition cell proliferation is better than the short ability of cell proliferation of platelet derived growth factor (PDGF-BB), and bone morphogenesis protein-7 (BMP-7) is relevant with bone morphogenesis protein-7 (BMP-7) dosage to the inhibition of platelet derived growth factor (PDGF-BB).Hatch after 2 days, compared with only processing with 20ng/mL platelet derived growth factor (PDGF-BB), depressing proliferation rate when bone morphogenesis protein-7 (BMP-7) concentration is 20ng/ml and 200ng/ml is respectively 17% and 24%.
It may be because it is that one lures bone morphogenetic protein(BMP) that bone morphogenesis protein-7 (BMP-7) suppresses cell proliferation, therefore can block mescenchymal stem cell (MSC) propagation, and Cell differentiation inducing activity is osteoblast.Cell cycle experiment shows, compared with matched group, uses after 20ng/mL platelet derived growth factor (PDGF-BB) processing, and G0, the cell number of G1 phase obviously reduce, the number showed increased of S, G2 and M phase.On the contrary, with 100ng/ml bone morphogenesis protein-7 (BMP-7) or simultaneously containing after 100ng/ml bone morphogenesis protein-7 (BMP-7) and 20ng/ml platelet derived growth factor (PDGF-BB) processing, the cell number showed increased of G0, G1 phase, S, G2 and the number of M phase obviously reduce.Fig. 2 shows, uses separately bone morphogenesis protein-7 (BMP-7) or uses not obviously difference with platelet derived growth factor (PDGF-BB) simultaneously.Experimental result shows, separately or combine with platelet derived growth factor (PDGF-BB) and use bone morphogenesis protein-7 (BMP-7) that cell growth cycle was blocked in the G1 phase.
2.2 lure the factorial analysis of mescenchymal stem cell between bone marrow (MSC) Osteoblast Differentiation
Detect somatomedin (Growth factors, GFs) to the active impact of alkali phosphatase (ALP) by alkali phosphatase (Alkaline phosphatase, the ALP)-time graph after the different sample treatment cells of quantitative biochemical colorimetric method for determining.As shown in Figure 3, process cell after 4 weeks with platelet derived growth factor (PDGF-BB), alkali phosphatase (ALP) is active not to be strengthened, and process after 2 weeks with bone morphogenesis protein-7 (BMP-7), alkali phosphatase (ALP) activity significantly strengthens, and after detection finishes, keeps this potentiation.Compared with independent use bone morphogenesis protein-7 (BMP-7), add after P platelet derived growth factor (PDGF-BB), alkali phosphatase (ALP) is active reduces approximately 4 times.
Extract the calcium in the calcium mineral obtaining with 0.6N hydrochloric acid (HCl) after different sample treatment cells, and measure calcium content to carry out detection by quantitative.As shown in Figure 4, in the experimental group of matched group and platelet derived growth factor (PDGF-BB) processing, calcium concentration is all lower, with bone morphogenesis protein-7 (BMP-7) or dexamethasone (dexamethasone, DEX) process, or after platelet derived growth factor (PDGF-BB) and bone morphogenesis protein-7 (BMP-7) process simultaneously, calcium concentration is higher, and increases in time and increase.Bone morphogenesis protein-7 (BMP-7) calcium concentration after treatment is suitable with DEX calcium concentration after treatment, but platelet derived growth factor (PDGF-BB) and bone morphogenesis protein-7 (BMP-7) while calcium concentration after treatment are lower than its half value.Experimental result shows, if release simultaneously, platelet derived growth factor (PDGF-BB) can be blocked the mineralization of bone morphogenesis protein-7 (BMP-7).
2.1 and 2.2 result of study demonstration, these two kinds of somatomedin mechanisms of action are different with the stage of working.While using simultaneously, if can not well control release time, have and influence each other, even inhibitory action, can not make both to reach good effect.
2.3 control the research that discharges platelet derived growth factor (PDGF-BB) and bone morphogenesis protein-7 (BMP-7)
As shown in Fig. 5-6, first the bone morphogenesis protein-7 (BMP-7) being embedded in shell discharges, and continues a large amount of release and can slowly discharge 10 days after 6 days.Compare, be embedded in platelet derived growth factor (PDGF-BB) in shell and discharge by S type curve, within initial 10 days, burst size is less, and next 2 weeks, burst size increased gradually and reaches maximum.If the loading position of two kinds of somatomedin of exchange, will keep its release mode, the albumen being embedded in shell first discharges, and the albumen being embedded in core discharges afterwards.
As shown in Fig. 5,7, when fixing stratum nucleare material is PLLA(viscosity 1.0dL/g) time, the core-shell particles that in change Shell Materials composite (PLGA) prepared by the proportioning of PLLA:PGA discharges somatomedin (BMP7, Fig. 5; PDGF-BB, Fig. 7) speed difference, in the time that the ratio of PLLA in PLGA improves, discharge the rate reduction of somatomedin.As shown in Fig. 6,8, when set casing layer material is PLGA(PLLA:PGA mass ratio=50:50) time, core-shell particles prepared by the viscosity of change stratum nucleare material PLLA discharges somatomedin (PDGF-BB, Fig. 5; BMP-7, Fig. 7) speed difference, in the time that the viscosity of PLLA increases, discharge the rate reduction of somatomedin.When fixing stratum nucleare material is PLLA(viscosity 1.0dL/g) time, in shell composite, when PLLA:PGA mass ratio=50:50, the priority that can realize both somatomedin discharges, and effect is better.
Measure the impact of various micro-sphere structures on somatomedin envelop rate (EE) in the application and comparative example, the results are shown in Table 1, and record different release profiles, see Fig. 9-10.
In table 1, contrast U and P are known, and nucleocapsid structure has significantly improved the envelop rate of somatomedin in core, and in contrast U and the known shell of S1/S2, the envelop rate of somatomedin is unchanged.The release result demonstration initial stage of common monolayer microsphere (U group) presents explosion type and discharges, and in initial 6 days, the burst size of platelet derived growth factor (PDGF-BB) is higher than 60%, and the burst size of bone morphogenesis protein-7 (BMP-7) reaches 50%.The following days platelet derived growth factor (PDGF-BB) and bone morphogenesis protein-7 (BMP-7) are linear and discharge, until the 30th day time, the burst size of two kinds of somatomedin reaches 85%.From this respect, in U group, two kinds of somatomedin are parallel release mode.Compared with simple monolayer microsphere, the double-deck micro-sphere structure complexity of nucleocapsid, can realize multiple controlled release pattern.
For example, the linearity that in nucleocapsid, all the PB group of embedding platelet derived growth factor (PDGF-BB) can realize platelet derived growth factor (PDGF-BB) discharges (the small burst of ignoring the initial period discharges).Same, the burst of ignoring the initial period discharges, and in nucleocapsid, all the B of embedding bone BMP-7 organizes the linearity release that also can realize BMP-7.P group core-shell particles embedding platelet derived growth factor (PDGF-BB) and bone morphogenesis protein-7 (BMP-7) simultaneously, compared with the monolayer microsphere U group of embedding equivalent somatomedin, the rate of release of two kinds of somatomedin is all slow, particularly at first 2 weeks (seeing Fig. 9-10).It is fast that P group discharges the speed ratio PB group of platelet derived growth factor (PDGF-BB), and possible cause is, in P group, nucleocapsid microsphere set has suffered the protein content (seeing Fig. 9-10) of 2 times of the same areas (core or shell) in PB group.
If two kinds of somatomedin are embedded in respectively in core-shell particles, the control that can realize two kinds of somatomedin discharges.As shown in Fig. 9,10, embedding platelet derived growth factor (PDGF-BB) and the S1 group of bone morphogenesis protein-7 (BMP-7) in core and shell respectively, at the 1st week, the burst size of bone morphogenesis protein-7 (BMP-7) reaches 85%, and the burst size of platelet derived growth factor (PDGF-BB) is only 15%, platelet derived growth factor (PDGF-BB) rate of release is accelerated afterwards, and after 3 weeks, burst size reaches 85%.In like manner, S2 group is after 1 week, and platelet derived growth factor (PDGF-BB) burst size reaches 95%, and bone morphogenesis protein-7 (BMP-7) burst size is only 10%, and bone morphogenesis protein-7 (BMP-7) rate of release is accelerated afterwards, and after 3 weeks, burst size reaches 88%.
The definition of each group sample and albumen envelop rate in table 1 experiment
The 0th day time, inoculate certain density mescenchymal stem cell (MSC) cell in culture dish.The platelet derived growth factor (PDGF-BB) discharging from core-shell particles stimulates mescenchymal stem cell (MSC) propagation to reach after the state of being paved with, and discharges the short cell Osteoblast Differentiation of having cultivated of bone morphogenesis protein-7 (BMP-7).Figure 11 shown after 4 weeks, blank, alkali phosphatase (ALP) activity level of matched group 1,2 and PB group is suitable, and alkali phosphatase (ALP) intensity of other groups is all higher than matched group (P<0.05).In U group, the rate of release of two kinds of somatomedin is greater than P group, but they facilitate bone differentiation capability suitable, and be all less than B group and S2 group.S1 and S2 are except the release order of platelet derived growth factor (PDGF-BB) and bone morphogenesis protein-7 (BMP-7) is different, and release mode is identical.Facilitate bone Analytical Chemical Experiment to show, the release order of platelet derived growth factor (PDGF-BB) and bone morphogenesis protein-7 (BMP-7) should strictly be controlled.Alkali phosphatase (ALP) activity level of S2 group is 6 times (platelet derived growth factor (PDGF-BB) is identical with the total content of bone morphogenesis protein-7 (BMP-7)) of U and P group.
Measure not calcium content on the same group after 4 weeks.From Figure 12, can obtain, matched group, the calcium content of blank group 1,2 is little, and U, PB, B, P, the calcium content in S1 and S2 group is more, and S2 group calcium content is than B group high (p<0.05).U, the value of P and S1 group is 1/4 of S2 group.Experimental result shows, when platelet derived growth factor (PDGF-BB) and bone morphogenesis protein-7 (BMP-7) discharge simultaneously or bone morphogenesis protein-7 (BMP-7) discharges prior to platelet derived growth factor (PDGF-BB), what platelet derived growth factor (PDGF-BB) can blocking-up bone morphogenesis protein-7 (BMP-7) facilitates bone differentiation.
Comprehensive all above evidences, the optimal mode of inducing mescenchymal stem cell Osteoblast Differentiation between bone marrow under low cell density condition of culture is first to discharge PDGF-BB, then discharges BMP-7(group S2).
Claims (10)
1. a nucleocapsid structure microsphere for the two somatomedin of parcel, comprises nucleocapsid spherical structure, it is characterized in that, described core is polylactic acid, and polylactic acid viscosity is 0.5-2.0dL/g; Shell is the composite of polylactic acid and polyglycolic acid composition, and in described composite, the mass ratio of polylactic acid and polyglycolic acid is: 75-50:25-50; In described core and shell, be enclosed with respectively a kind of somatomedin, described somatomedin is BMP-7 or PDGF-BB.
2. a nucleocapsid structure microsphere for the two somatomedin of parcel, is characterized in that, the somatomedin of described core parcel is BMP-7, and the somatomedin of described shell parcel is PDGF-BB.
3. according to the nucleocapsid structure microsphere of the two somatomedin of parcel described in claim 1 or 2, it is characterized in that, the external diameter of described nucleocapsid structure microsphere is 18-22 micron, the mass ratio of described core and shell is: 1:3-4, the mass percent of somatomedin in core is 0.002%-0.005%, and the mass percent of somatomedin in shell is 0.0008%-0.00125%.
4. according to the nucleocapsid structure microsphere of the two somatomedin of parcel described in claim 1 or 2, it is characterized in that, described polylactic acid viscosity is 1.0dL/g.
5. according to the nucleocapsid structure microsphere of the two somatomedin of parcel described in claim 1 or 2, it is characterized in that, the mass ratio of described polylactic acid and polyglycolic acid is: 50:50.
6. according to the nucleocapsid structure microsphere of the two somatomedin of the described parcel of one of claim 1-5, it is characterized in that, prepare by the following method:
(1) PDGF-BB is dissolved in to deionized water and makes the water I that concentration is 1 μ g/ml-100 μ g/ml, composite is dissolved in to dichloromethane and makes the organic facies I that quality percent by volume is 8%-12%, water I and organic facies I mix rear ultrasonic emulsification for 1:8-12 by volume, obtain emulsion I; By same method, BMP-7 is dissolved in to deionized water and makes the water II that concentration is 1 μ g/ml-100 μ g/ml, polylactic acid is dissolved in to ethyl acetate and prepares the organic facies II that quality percent by volume is 2%-5%, water II and organic facies II mix rear ultrasonic emulsification for 1:8-12 by volume, obtain emulsion II;
(2) adopt coaxial electrostatic spray technique, emulsion I and emulsion II are sprayed into respectively in the outer pin and interior pin of coaxial needles of electrostatic atomizer, in controlling, in pin, the flow of material is 1.0-1.2ml/h, the flow of controlling material in outer pin is 1.5-2.0ml/h, 10cm-12cm place below coaxial needles, the microsphere of preparing with the culture dish reception of filling dehydrated alcohol, then washing, centrifugal again, lyophilization.
7. the nucleocapsid structure microsphere that wraps up according to claim 6 two somatomedin, is characterized in that, in described water I, the concentration of PDGF-BB is 5 μ g/ml-20 μ g/ml, and in described organic facies I, the quality percent by volume of composite is 10%; In described water II, the concentration of BMP-7 is 5 μ g/ml-20 μ g/ml, and in described organic facies II, the quality percent by volume of composite is 3%.
According to the nucleocapsid structure microsphere of the two somatomedin of the described parcel of one of claim 1-7 in the application of preparing in inducing cell propagation medicine.
9. the application between the bone marrow of cultivating at the low cell density of preparation induction according to the nucleocapsid structure microsphere of the two somatomedin of the described parcel of one of claim 1-7 in mescenchymal stem cell Osteoblast Differentiation medicine.
10. the application in preparation treatment fracture and injury medicine according to the nucleocapsid structure microsphere of the two somatomedin of the described parcel of one of claim 1-7.
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| HEMIN NIE等: "Core/shell microspheres via coaxial electrohydrodynamic atomization for sequential and parallel release of drugs", 《JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A》, vol. 95, no. 3, 1 December 2010 (2010-12-01), pages 709 - 716 * |
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