CN104189954B - A kind of in-situ solidifying tissue engineering bracket and preparation method thereof - Google Patents
A kind of in-situ solidifying tissue engineering bracket and preparation method thereof Download PDFInfo
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- CN104189954B CN104189954B CN201410483564.5A CN201410483564A CN104189954B CN 104189954 B CN104189954 B CN 104189954B CN 201410483564 A CN201410483564 A CN 201410483564A CN 104189954 B CN104189954 B CN 104189954B
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Abstract
The invention provides a kind of in-situ solidifying tissue engineering bracket, by polylactide and biodegradable microspheres, the in-situ solidifying in the presence of polar organic solvent is formed.The in-situ solidifying tissue engineering bracket that the present invention is provided is using biodegradable microspheres as pore-foaming agent, microballoon in in-situ solidifying tissue engineering bracket is gradually degraded, its pore structure is gradually formed, porosity gradually increases, internal gradient pore from outside to inside can be realized so as to which hole is formed to be grown into tissue and matched.The in-situ solidifying initial stage of the in-situ solidifying tissue engineering bracket provided in the present invention, due to the presence of the undegradable pore-foaming agent in part so that the initial strength of the in-situ solidifying tissue engineering bracket that the present invention is provided is significantly improved.In addition, the in-situ solidifying tissue engineering bracket that the present invention is provided is with biodegradable microspheres as pore-foaming agent, good biocompatibility, it is to avoid by the hypertonic to histiocytic damage of the particles such as salt.
Description
Technical field
The invention belongs to biomedicine field, more particularly to a kind of in-situ solidifying tissue engineering bracket and preparation method thereof.
Background technology
According to statistics, the Cranial defect patient that China is caused because of disease, wound, tumour etc. every year is more than 1,000,000, and becomes in rising
Gesture.Autologous bone transplanting and homogeneous allogenic bone transplantation are the main methods for treating Cranial defect at present.However, autologous bone limited source,
And cause new defect, application to be restricted to donor site.Homogeneous allogenic bone self-bone grafting ability is poor compared with autologous bone, and there is row
Scold the danger of reaction and transmission.In recent years, increasing Inorganic synthese material or organic synthesis macromolecular material quilt
Research and development is used as tissue engineering bracket or bone renovating material, and is progressively applied to clinic.
And injectable organizational project biological support can solve the problems, such as to appear above.Way of the bone renovating material by injection
Footpath reaches Cranial defect position, is filled up completely with rapidly defect, realizes matching completely for graft materials and host's Cranial defect, can have
Effect stimulates the growth of stress of bone tissue.This non-invasive procedures also avoid the damage again of surrounding soft tissue, and operating time is short, into
This is low, it is easier to is received by patient, is conducive to the promotion and application of medical new material.
Shaped in situ graft (in situ forming implants, ISI) is a kind of injectable bio-medical material
Material, is that high molecular polymer is dissolved in organic solvent that can be miscible with water, after the mixture for obtaining is injected in tissue, is had
Machine solvent is replaced by the water in tissue rapidly, the solidification of high molecular polymer shaped in situ.
CWRU Krebs in 2009 etc. is dissolved in PLGA (polylactide-co-glycolide) in tetraethylene-glycol, with
Sodium salt particle prepares the injectable bone renovating material of porous as pore-foaming agent.Germany scientist Schloegl passes through within 2012
The different proportion optimization support of adjustment PLGA, α-TCP, organic solvent and sodium salt (pore-foaming agent), through the timbering material hole for optimizing
Gap rate can reach 81.2%-91.5%.But, using sodium salt as pore-foaming agent, sodium salt enters group with support injection to above-mentioned technology
After knitting, initial mechanical properties are less, only 0.285MPa, it is impossible to meet the reparation needs of weight bearing area.
The content of the invention
It is an object of the invention to provide a kind of in-situ solidifying tissue engineering bracket and preparation method thereof, what the present invention was provided
In-situ solidifying tissue engineering bracket has higher initial mechanical properties, and its preparation method is simple.
The present invention provides a kind of in-situ solidifying tissue engineering bracket, by polylactide-co-glycolide and biodegradable microspheres
In the presence of polar organic solvent, in-situ solidifying is formed.
Preferably, the biodegradable microspheres are gelatine microsphere, polylactide-co-glycolide microballoon, alginate microsphere
With one or more in chitosan microball.
Preferably, the particle diameter of the biodegradable microspheres is 100～450 μm.
Preferably, the number-average molecular weight of the polylactide-co-glycolide is 50000～200000.
Preferably, the polylactide-co-glycolide and the mass ratio of biodegradable microspheres are (1～9)：(9～1).
Preferably, the polar organic solvent is 1-Methyl-2-Pyrrolidone, dimethyl sulfoxide (DMSO), tetraethylene glycol and acetic acid second
One or more in ester.
Preferably, the porosity of the in-situ solidifying tissue engineering bracket is 70～95%.
Preferably, the in-situ solidifying tissue engineering bracket also includes hydroxyapatite.
Preferably, the biodegradable microspheres are the biodegradable microspheres for supporting activated protein.
The present invention provides a kind of preparation method of in-situ solidifying tissue engineering bracket, comprises the following steps：
Polylactide-co-glycolide, biodegradable microspheres and polar organic solvent are mixed, Jing in-situ solidifyings obtain original
Position solidification tissue engineering bracket.
The invention provides a kind of in-situ solidifying tissue engineering bracket, micro- by polylactide-co-glycolide and biodegradable
Ball in-situ solidifying in the presence of polar organic solvent is formed.The in-situ solidifying tissue engineering bracket that the present invention is provided can with biology
, used as pore-foaming agent, by the in-situ solidifying pore-forming tissue injection to position is repaired in vivo, polar organic solvent is by body for degraded microballoon
Water displacement in liquid, it is fast curing-formed in original position.As the degradation rate of the biodegradable microspheres is compared with polylactide-second
Lactide is fast, pore-forming of gradually degrading, and guiding tissue grows into, and final material is degradable to be repaired tissue substitute, reaches reparation purpose.
Microballoon in the in-situ solidifying tissue engineering bracket that the present invention is provided gradually is degraded, and its pore structure is gradually formed, and porosity is gradually
Increase, it is possible to achieve gradient pore from outside to inside in vivo so as to which hole is formed to be grown into tissue and matched.There is provided in the present invention
The in-situ solidifying initial stage of in-situ solidifying tissue engineering bracket, due to the presence of the undegradable pore-foaming agent in part so that the present invention is carried
For the initial strength of in-situ solidifying tissue engineering bracket significantly improve.Also, in-situ solidifying organizational project of the present invention
The preparation method of frame is simple.
In addition, the in-situ solidifying tissue engineering bracket that the present invention is provided is with biodegradable microspheres as pore-foaming agent, biofacies
Capacitive is good, it is to avoid by the hypertonic to histiocytic damage of the particles such as salt.
Further, the biodegradable microspheres are used as can also support growth factor isoreactivity egg while pore-foaming agent
White or other medicines, pore-foaming agent microballoon can effective protection protein drug activity, it is to avoid active growth factor and organic solvent
Directly contact and loss of activity.
Description of the drawings
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
Accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are only this
Inventive embodiment, for those of ordinary skill in the art, on the premise of not paying creative work, can be with basis
The accompanying drawing of offer obtains other accompanying drawings.
The preparation process schematic diagram of the in-situ solidifying tissue engineering bracket that Fig. 1 is provided for the present invention；
Fig. 2 is the mechanical strength figure of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 1～9 is obtained；
Fig. 3 is the scanning electron microscope (SEM) photograph of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 1 is obtained.
Specific embodiment
The invention provides a kind of in-situ solidifying tissue engineering bracket, micro- by polylactide-co-glycolide and biodegradable
Ball in-situ solidifying in the presence of polar organic solvent is formed.
The in-situ solidifying tissue engineering bracket that the present invention is provided has higher initial mechanical properties, preferable bio-compatible
The advantages of ability of property and protein medicine.
The in-situ solidifying tissue engineering bracket that the present invention is provided is by polylactide-co-glycolide and biodegradable microspheres in pole
In the presence of property organic solvent, in-situ solidifying is formed.In the present invention, the porosity of the in-situ solidifying tissue engineering bracket is excellent
Elect 70～95%, more preferably 75～90%, most preferably 80～85% as.
In the present invention, the number-average molecular weight of the polylactide-co-glycolide (PLGA) is preferably 50000～200000,
More preferably 55000～195000, most preferably 60000～190000, source of the present invention to the polylactide-co-glycolide
Without special restriction, the commercial goods of the polylactide-co-glycolide can be adopted, also can be known according to those skilled in the art
The technical scheme for preparing polylactide-co-glycolide voluntarily prepare.
In the present invention, the biodegradable microspheres are preferably gelatine microsphere, polylactide-co-glycolide microballoon, marine alga
One or more in hydrochlorate microballoon and chitosan microball, more preferably gelatine microsphere, alginate microsphere and chitosan microball
In one or more, most preferably gelatine microsphere；The particle diameter of the biodegradable microspheres is preferably 100～450 μm, more excellent
Elect 150～400 μm as, most preferably 200～350 μm；The mass ratio of the polylactide-co-glycolide and biodegradable microspheres
Preferably (1～9)：(9～1), more preferably (1.5～8.5)：(8.5～1.5), most preferably (2～8)：(8～2).In order to
Make described in-situ solidifying tissue engineering bracket that there is certain feature, in the present invention, the biodegradable microspheres are excellent
Elect the biodegradable microspheres for being supported with activated protein as, the present invention does not have special limit to the activated protein species and consumption
System, adopts appropriate activated protein according to actual demand.Such as, bone morphogenetic protein, vascular endothelial growth can be adopted
One or more in the factor and insulin-like growth factor.
The present invention does not have special restriction to the source of the biodegradable microspheres, and in the present invention, the biology can
Degraded microballoon preferably follows the steps below preparation：
A) Biodegradable material is mixed with water, Biodegradable material solution is obtained；
B) by atoleine, sorbitol anhydride oleate and step A) the biodegradable microspheres solution that obtains mixes
Close, obtain mixture；
C) by step B) mixture that obtains filtered, and obtains biodegradable microspheres.
Biodegradable material is mixed by the present invention with water, obtains Biodegradable material solution, and the present invention is preferably by institute
State Biodegradable material to mix with deionized water, obtain Biodegradable material solution.In the present invention, the biology can drop
One or more in solution material preferably gelatin, polylactide-co-glycolide, alginate and shitosan, more preferably gelatin,
One or more in alginate and shitosan, most preferably gelatin；The temperature of the mixing is preferably 30～40 DEG C, more excellent
Elect 35～38 DEG C as, most preferably 37 DEG C；The mass concentration of the Biodegradable material solution preferably 15～25%, it is more excellent
Elect 18～22%, most preferably 19～20% as.
After obtaining Biodegradable material solution, the Biodegradable material solution is preferably entered horizontal high voltage and is gone out by the present invention
Bacterium, the autoclaved pressure are preferably 100～150kPa, more preferably 120 kPa；The autoclaved time is preferred
For 20～30 minutes, more preferably 25 minutes.
In order that the biodegradable microspheres have certain feature, the present invention is preferably by the biodegradable for obtaining
Material solution is mixed with activated protein, obtains being supported with the Biodegradable material solution of activated protein.In the present invention, it is described
The species and consumption of activated protein are consistent with the species and consumption of activated protein in above-mentioned technical proposal, will not be described here.This
Mixed method of the invention to the Biodegradable material solution with activated protein does not have special restriction, using art technology
Mixed method known to personnel.
After obtaining Biodegradable material solution, the present invention by atoleine, sorbitol anhydride oleate (Span-80) and
The Biodegradable material solution mixing, obtains mixture.The atoleine and Span-80 are preferably mixed by the present invention,
Then mix with the Biodegradable material solution again, obtain mixture.The present invention is to the atoleine and Span-80
Source does not have special restriction, using the atoleine and the commercial goods of Span-80.In the present invention, the liquid
The volume ratio of body paraffin and Span-80 is preferably (350～500)：1, more preferably (400～450)：1, the present invention is preferably in water
Under the conditions of bath, the mixing of the atoleine and Span-80 is carried out, and the temperature of the water-bath is preferably 30～40 DEG C, more preferably
For 35～38 DEG C, most preferably 37 DEG C, the present invention does not have special restriction to the time of the water-bath, can make the liquid stone
Wax and Span-80 are well mixed.
After completing the mixing of the atoleine and Span-80, the present invention is preferably by the Biodegradable material solution
In atoleine and the mixture of Span-80 that addition is obtained, it is stirred, obtains mixture.The present invention more preferably will be described
During Biodegradable material solution adds the mixture of the atoleine and Span-80 for obtaining, the first stirring is carried out, complete described
After first stirring, the product of the first stirring is carried out into the second stirring under conditions of ice bath, mixture is obtained.In the present invention,
The Biodegradable material solution is preferably 1 with the volume ratio of atoleine：(1～3), more preferably 1:(1.5～2.5),
Most preferably 1：2；The time of first stirring is preferably 3～10min, more preferably 4～9min；The speed of first stirring
Degree is preferably 100～300rpm, more preferably 200rpm；The temperature of first stirring is preferably 30～40 DEG C, more preferably
35～38 DEG C, most preferably 37 DEG C；The temperature of second stirring is preferably 0～4 DEG C, more preferably 2 DEG C；Second stirring
Time be preferably 50～80min, more preferably 60～70min；The speed of second stirring is preferably 100～300rpm,
More preferably 200rpm.
After obtaining mixture, the mixture is filtered by the present invention, obtains biodegradable microspheres.It is of the invention preferred
Filtered using filter screen, the aperture of the filter screen is preferably 100～450 μm, more preferably 200～400 μm, most preferably
300μm.The present invention does not have special restriction to the method for the filtration, using the technology of filtration well known to those skilled in the art
Scheme.
After completing the filtration, the biodegradable microspheres being filtrated to get preferably are dried by the present invention, obtain drying
Biodegradable microspheres.The biodegradable microspheres being filtrated to get preferably are carried out freeze-drying by the present invention, obtain drying
Biodegradable microspheres.In the present invention, the cryodesiccated temperature is preferably -80～-20 DEG C, and more preferably -50～-30
DEG C, most preferably -45～-35 DEG C；The cryodesiccated time is preferably 18～30 hours, more preferably 20～26 hours.
After completing the drying, dry biodegradable microspheres are preferably cleaned by the present invention, after being cleaned
Biodegradable microspheres.Present invention preferably employs absolute alcohol is cleaned to the biodegradable microspheres of the drying, obtain
Biodegradable microspheres after cleaning.The present invention does not have special restriction to the source of the absolute alcohol and consumption, can be by
The biodegradable microspheres of the drying are cleaned up.The present invention does not have special restriction, energy to the number of times of the cleaning
It is enough that the biodegradable microspheres of the drying are cleaned up.
In the present invention, the polar organic solvent is preferably 1-Methyl-2-Pyrrolidone (NMP), dimethyl sulfoxide (DMSO)
(DMSO), one or more in tetraethylene glycol and ethyl acetate, more preferably 1-Methyl-2-Pyrrolidone and/or dimethyl are sub-
Sulfone, most preferably 1-Methyl-2-Pyrrolidone, the polylactide-co-glycolide are preferably with the mass ratio of polar organic solvent
1：(2.5～5), more preferably 1：(2.8～4.5), most preferably 1：(3～4).
In the present invention, in order that the in-situ solidifying tissue engineering bracket that the present invention is provided has in bone tissue reparation more preferably
Application, the in-situ solidifying tissue engineering bracket preferably also include hydroxyapatite (HA), hydroxyapatite is skeleton
Main component, will in Hydroxapatite implant body can induce bone tissue growth.In the present invention, the matter of the HA and PLGA
Amount ratio preferably 1：(10～1.5), more preferably 1：(9～2), most preferably 1：(8～3).Source of the present invention to the HA
Without special restriction, the HA commonly used using those skilled in the art.
Present invention also offers a kind of preparation method of in-situ solidifying tissue engineering bracket, comprises the following steps：
Polylactide-co-glycolide, biodegradable microspheres and polar organic solvent are mixed, Jing in-situ solidifyings obtain original
Position solidification tissue engineering bracket.
Polylactide-co-glycolide (PLGA) is preferably mixed by the present invention with polar organic solvent, obtains PLGA solution, will be raw
Biodegradable microballoon is mixed with described PLGA solution, and Jing in-situ solidifyings obtain in-situ solidifying tissue engineering bracket.In order that
To in-situ solidifying tissue engineering bracket can be applied to bone tissue reparation, the present invention preferably by PLGA, polar organic solvent and
Hydroxyapatite (HA) mixes, and obtains PLGA solution, biodegradable microspheres are mixed with described PLGA solution, and Jing is in situ solid
Change, obtain in-situ solidifying tissue engineering bracket.In the present invention, the source of the PLGA, polar organic solvent and HA and consumption
It is consistent with the source of PLGA, polar organic solvent and HA and consumption in above-mentioned technical proposal, will not be described here.In the present invention
In, the temperature of the mixing of the PLGA, polar organic solvent and HA is preferably 60～80 DEG C, more preferably 65～75 DEG C.This
The bright mixing to the PLGA, polar organic solvent and HA does not have a special restriction, preferred pair of the present invention its carry out the 3rd stirring,
The mixing of the PLGA, polar organic solvent and HA is completed, the present invention does not have special restriction to the method for the described 3rd stirring,
Using the technical scheme of stirring well known to those skilled in the art.
After obtaining PLGA solution, the biodegradable microspheres are mixed by the present invention with the PLGA solution, obtain noting
The in-situ solidifying tissue engineering bracket penetrated, the injectable in-situ solidifying tissue engineering bracket Jing in-situ solidifyings, obtains original position
Solidification tissue engineering bracket.In the present invention, the source of the biodegradable microspheres and consumption are raw with above-mentioned technical proposal
The source of Biodegradable microballoon is consistent with consumption, will not be described here.The present invention to the biodegradable microspheres with it is described
The mixed method of PLGA solution does not have a special restriction, preferred pair of the present invention its carry out the 4th stirring, completing the biology can drop
Solve the mixing of microballoon and the PLGA solution.The present invention does not have special restriction to the method for the described 4th stirring, using ability
The biodegradable microspheres are stirred by the stirring technique scheme that field technique personnel commonly use with the PLGA solution.
After obtaining injectable in-situ solidifying tissue engineering bracket, Injectable in-situ solidification is preferably organized by the present invention
Engineering rack is injected in the aqueous solution, carries out in-situ solidifying, obtains in-situ solidifying tissue engineering bracket.The present invention is to the injection
Method there is no special restriction, the technical scheme of the injection commonly used using those skilled in the art.The present invention is to described
The consumption of injectable in-situ solidifying tissue engineering bracket does not have special restriction, using appropriate consumption is according to the actual requirements
Can.
The preparation process schematic diagram of the in-situ solidifying tissue engineering bracket provided for the present invention referring to Fig. 1, Fig. 1.1 is by life
During Biodegradable microballoon adds PLGA solution, 2 is the injectable in-situ solidifying tissue engineering bracket for obtaining, and 3 is by injectable
In-situ solidifying tissue engineering bracket be injected to the aqueous solution in carry out in-situ solidifying.As seen from Figure 1, the original that the present invention is provided
Position solidification tissue engineering bracket preparation method is simple, it is easy to operate.
The present invention determines the in-situ solidifying tissue for obtaining according to GT/T 1041-92 standards using the method for direct weighting
Mechanical strength of the engineering rack in solidification 10min.As a result show, the present invention provide in-situ solidifying tissue engineering bracket it is initial
Mechanical strength can reach 2.8MPa, and the initial mechanical properties 0.285MPa of more existing in-situ solidifying tissue engineering bracket is improved
10 times.
The present invention determines the in-situ solidifying organizational project for obtaining according to liquid displacement technique (liquid displacement)
The porosity of support, as a result shows, the porosity of the in-situ solidifying tissue engineering bracket that the present invention is provided is obtained up to 92%
Brace aperture rate is higher, is conducive to the growth in situ of bone tissue.
The invention provides a kind of in-situ solidifying tissue engineering bracket, micro- by polylactide-co-glycolide and biodegradable
Ball in-situ solidifying in the presence of polar organic solvent is formed.The in-situ solidifying tissue engineering bracket that the present invention is provided can with biology
, used as pore-foaming agent, by the in-situ solidifying pore-forming tissue injection to position is repaired in vivo, polar organic solvent is by body for degraded microballoon
Water displacement in liquid, it is fast curing-formed in original position.As the degradation rate of the biodegradable microspheres is compared with polylactide-second
Lactide is fast, pore-forming of gradually degrading, and guiding tissue grows into, and final material is degradable to be repaired tissue substitute, reaches reparation purpose.
Microballoon in the in-situ solidifying tissue engineering bracket that the present invention is provided gradually is degraded, and its pore structure is gradually formed, and porosity is gradually
Increase, it is possible to achieve gradient pore from outside to inside in vivo so as to which hole is formed to be grown into tissue and matched.There is provided in the present invention
The in-situ solidifying initial stage of in-situ solidifying tissue engineering bracket, due to the presence of the undegradable pore-foaming agent in part so that the present invention is carried
For the initial strength of in-situ solidifying tissue engineering bracket significantly improve.In addition, the in-situ solidifying organizational project that the present invention is provided
Support with biodegradable microspheres as pore-foaming agent, good biocompatibility, it is to avoid by the hypertonic to histiocytic of the particles such as salt
Damage.
Further, the biodegradable microspheres are used as can also support growth factor isoreactivity egg while pore-foaming agent
White or other medicines, pore-foaming agent microballoon can effective protection protein drug activity, it is to avoid active growth factor and organic solvent
Directly contact and loss of activity.
In order to further illustrate the present invention, a kind of in-situ solidifying organizational project that with reference to embodiments present invention is provided
Support and preparation method thereof is described in detail, but can not be understood as limiting the scope of the present invention.
Embodiment 1
Gelatin is prepared in 37 DEG C of deionized water the gelatin solution for obtaining that mass concentration is 25%, Ran Hou
Under 120kPa after autoclaving, bone morphogenetic protein (BMP-2) is added in gelatin solution, the concentration for obtaining BMP-2 is
The gelatin solution of 500ng/mL.
The Span-80 of 20mL atoleines and 50 μ L is added in beaker, is stirred under 37 DEG C of water bath conditions, is taken
10mL gelatin solutions are added thereto, and 10min is stirred under 200rpm rotating speeds, then keep rotating speed constant, are transferred to 4 DEG C of ice bath
Under the conditions of, 1h is persistently stirred, mixture is obtained.
Using the strainer filtering that aperture is 300 μm, by the microballoon being filtrated to get at -50 DEG C freeze-drying 24h.
With the gelatine microsphere after the quick cleaning-drying repeatedly of absolute alcohol, that is, obtain gelatine microsphere.
At 80 DEG C, 3gPLGA is dissolved in 10gNMP, adds 1gHA, stir, obtain PLGA solution.
5gPLGA solution is mixed with 5g gelatine microspheres, is stirred to homogeneous, obtain injectable in-situ solidifying organizational project
Support.Containing 10.3% PLGA in the injectable in-situ solidifying tissue engineering bracket, 3.7% HA, 50% gelatin are micro-
Ball and 36% NMP.
Injectable in-situ solidifying tissue engineering bracket is injected in the aqueous solution, in-situ solidifying is carried out, obtains in situ solid
Change tissue engineering bracket.
The present invention tests the mechanical strength of in-situ solidifying tissue engineering bracket that the present embodiment obtains in solidification 10min,
As a result as shown in Fig. 2 Fig. 2 is the mechanical strength figure of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 1～9 is obtained.
The present invention tests the porosity of the in-situ solidifying tissue engineering bracket that the present embodiment is obtained, and as a result shows, this reality
The porosity for applying the in-situ solidifying tissue engineering bracket that example is obtained is 92%.
The in-situ solidifying tissue engineering bracket that the present invention is obtained to the present embodiment has carried out sem test, as a result as schemed
Shown in 3, Fig. 3 is the scanning electron microscope (SEM) photograph of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 1 is obtained.In figure 3,1 and 2 are
The scanning electron microscope (SEM) photograph of the in-situ solidifying tissue engineering bracket section that the present embodiment is obtained；3 in-situ solidifyings obtained for the present embodiment
The scanning electron microscope (SEM) photograph of tissue engineering bracket surface.As seen from Figure 3, the in-situ solidifying tissue engineering bracket that the present embodiment is obtained
Porosity it is higher, and even pore distribution.
Embodiment 2
Gelatin is prepared in 37 DEG C of deionized water the gelatin solution for obtaining that mass concentration is 25%, Ran Hou
Under 123kPa after autoclaving, bone morphogenetic protein (BMP-2) is added in gelatin solution, the concentration for obtaining BMP-2 is
The gelatin solution of 500ng/mL.
The Span-80 of 20mL atoleines and 50 μ L is added in beaker, is stirred under 37 DEG C of water bath conditions, is taken
10mL gelatin solutions are added thereto, and 10min is stirred under 200rpm rotating speeds, then keep rotating speed constant, are transferred to 4 DEG C of ice bath
Under the conditions of, 1h is persistently stirred, mixture is obtained.
Using the strainer filtering that aperture is 300 μm, by the microballoon being filtrated to get at -40 DEG C freeze-drying 24h.
With the gelatine microsphere after the quick cleaning-drying repeatedly of absolute alcohol, that is, obtain gelatine microsphere.
At 80 DEG C, 3gPLGA is dissolved in 10gNMP, adds 1gHA, stir, obtain PLGA solution.
6gPLGA solution is mixed with 4g gelatine microspheres, is stirred to homogeneous, obtain injectable in-situ solidifying organizational project
Support.In the injectable in-situ solidifying tissue engineering bracket containing 13% PLGA, 4% HA, 40% gelatine microsphere and
43% NMP.
Injectable in-situ solidifying tissue engineering bracket is injected in the aqueous solution, in-situ solidifying is carried out, obtains in situ solid
Change tissue engineering bracket.
The present invention tests the mechanical strength of in-situ solidifying tissue engineering bracket that the present embodiment obtains in solidification 10min,
As a result as shown in Fig. 2 Fig. 2 is the mechanical strength figure of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 1～9 is obtained.
The present invention tests the porosity of the in-situ solidifying tissue engineering bracket that the present embodiment is obtained, and as a result shows, this reality
The porosity for applying the in-situ solidifying tissue engineering bracket that example is obtained is 86%.
Embodiment 3
Gelatin is prepared in 37 DEG C of deionized water the gelatin solution for obtaining that mass concentration is 25%, Ran Hou
Under 123kPa after autoclaving, bone morphogenetic protein (BMP-2) is added in gelatin solution, the concentration for obtaining BMP-2 is
The gelatin solution of 500ng/mL.
The Span-80 of 20mL atoleines and 50 μ L is added in beaker, is stirred under 37 DEG C of water bath conditions, is taken
10mL gelatin solutions are added thereto, and 10min is stirred under 200rpm rotating speeds, then keep rotating speed constant, are transferred to 4 DEG C of ice bath
Under the conditions of, 1h is persistently stirred, mixture is obtained.
Using the strainer filtering that aperture is 300 μm, by the microballoon being filtrated to get at -50 DEG C freeze-drying 24h.
With the gelatine microsphere after the quick cleaning-drying repeatedly of absolute alcohol, that is, obtain gelatine microsphere.
At 80 DEG C, 3gPLGA is dissolved in 10gNMP, adds 1gHA, stir, obtain PLGA solution.
7gPLGA solution is mixed with 3g gelatine microspheres, is stirred to homogeneous, obtain injectable in-situ solidifying organizational project
Support.In the injectable in-situ solidifying tissue engineering bracket containing 15% PLGA, 5% HA, 30% gelatine microsphere and
50% NMP.
Injectable in-situ solidifying tissue engineering bracket is injected in the aqueous solution, in-situ solidifying is carried out, obtains in situ solid
Change tissue engineering bracket.
The present invention tests the mechanical strength of in-situ solidifying tissue engineering bracket that the present embodiment obtains in solidification 10min,
As a result as shown in Fig. 2 Fig. 2 is the mechanical strength figure of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 1～9 is obtained.
The present invention tests the porosity of the in-situ solidifying tissue engineering bracket that the present embodiment is obtained, and as a result shows, this reality
The porosity for applying the in-situ solidifying tissue engineering bracket that example is obtained is 83%.
Embodiment 4
Gelatin is prepared in 37 DEG C of deionized water the gelatin solution for obtaining that mass concentration is 25%, Ran Hou
Under 123kPa after autoclaving, bone morphogenetic protein (BMP-2) is added in gelatin solution, the concentration for obtaining BMP-2 is
The gelatin solution of 500ng/mL.
The Span-80 of 20mL atoleines and 50 μ L is added in beaker, is stirred under 37 DEG C of water bath conditions, is taken
10mL gelatin solutions are added thereto, and 10min is stirred under 200rpm rotating speeds, then keep rotating speed constant, are transferred to 4 DEG C of ice bath
Under the conditions of, 1h is persistently stirred, mixture is obtained.
Using the strainer filtering that aperture is 300 μm, by the microballoon being filtrated to get at -50 DEG C freeze-drying 24h.
With the gelatine microsphere after the quick cleaning-drying repeatedly of absolute alcohol, that is, obtain gelatine microsphere.
At 80 DEG C, 3gPLGA is dissolved in 10gNMP, adds 2gHA, stir, obtain PLGA solution.
5gPLGA solution is mixed with 5g gelatine microspheres, is stirred to homogeneous, obtain injectable in-situ solidifying organizational project
Support.In the injectable in-situ solidifying tissue engineering bracket containing 10% PLGA, 7% HA, 50% gelatine microsphere and
33% NMP.
Injectable in-situ solidifying tissue engineering bracket is injected in the aqueous solution, in-situ solidifying is carried out, obtains in situ solid
Change tissue engineering bracket.
The present invention tests the mechanical strength of in-situ solidifying tissue engineering bracket that the present embodiment obtains in solidification 10min,
As a result as shown in Fig. 2 Fig. 2 is the mechanical strength figure of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 1～9 is obtained.
The present invention tests the porosity of the in-situ solidifying tissue engineering bracket that the present embodiment is obtained, and as a result shows, this reality
The porosity for applying the in-situ solidifying tissue engineering bracket that example is obtained is 87%.
Embodiment 5
Gelatin is prepared in 37 DEG C of deionized water the gelatin solution for obtaining that mass concentration is 25%, Ran Hou
Under 123kPa after autoclaving, bone morphogenetic protein (BMP-2) is added in gelatin solution, the concentration for obtaining BMP-2 is
The gelatin solution of 500ng/mL.
The Span-80 of 20mL atoleines and 50 μ L is added in beaker, is stirred under 37 DEG C of water bath conditions, is taken
10mL gelatin solutions are added thereto, and 10min is stirred under 200rpm rotating speeds, then keep rotating speed constant, are transferred to 4 DEG C of ice bath
Under the conditions of, 1h is persistently stirred, mixture is obtained.
Using the strainer filtering that aperture is 300 μm, by the microballoon being filtrated to get at -50 DEG C freeze-drying 24h.
With the gelatine microsphere after the quick cleaning-drying repeatedly of absolute alcohol, that is, obtain gelatine microsphere.
At 80 DEG C, 3gPLGA is dissolved in 10gNMP, adds 2gHA, stir, obtain PLGA solution.
6gPLGA solution is mixed with 4g gelatine microspheres, is stirred to homogeneous, obtain injectable in-situ solidifying organizational project
Support.In the injectable in-situ solidifying tissue engineering bracket containing 12% PLGA, 8% HA, 40% gelatine microsphere and
40% NMP.
Injectable in-situ solidifying tissue engineering bracket is injected in the aqueous solution, in-situ solidifying is carried out, obtains in situ solid
Change tissue engineering bracket.
The present invention tests the mechanical strength of in-situ solidifying tissue engineering bracket that the present embodiment obtains in solidification 10min,
As a result as shown in Fig. 2 Fig. 2 is the mechanical strength figure of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 1～9 is obtained.
The present invention tests the porosity of the in-situ solidifying tissue engineering bracket that the present embodiment is obtained, and as a result shows, this reality
The porosity for applying the in-situ solidifying tissue engineering bracket that example is obtained is 81%.
Embodiment 6
Gelatin is prepared in 37 DEG C of deionized water the gelatin solution for obtaining that mass concentration is 25%, Ran Hou
Under 123kPa after autoclaving, bone morphogenetic protein (BMP-2) is added in gelatin solution, the concentration for obtaining BMP-2 is
The gelatin solution of 500ng/mL.
The Span-80 of 20mL atoleines and 50 μ L is added in beaker, is stirred under 37 DEG C of water bath conditions, is taken
10mL gelatin solutions are added thereto, and 10min is stirred under 200rpm rotating speeds, then keep rotating speed constant, are transferred to 4 DEG C of ice bath
Under the conditions of, 1h is persistently stirred, mixture is obtained.
Using the strainer filtering that aperture is 300 μm, by the microballoon being filtrated to get at -50 DEG C freeze-drying 24h.
With the gelatine microsphere after the quick cleaning-drying repeatedly of absolute alcohol, that is, obtain gelatine microsphere.
At 80 DEG C, 3gPLGA is dissolved in 10gNMP, adds 2gHA, stir, obtain PLGA solution.
7gPLGA solution is mixed with 3g gelatine microspheres, is stirred to homogeneous, obtain injectable in-situ solidifying organizational project
Support.In the injectable in-situ solidifying tissue engineering bracket containing 14% PLGA, 9% HA, 30% gelatine microsphere and
47% NMP.
Injectable in-situ solidifying tissue engineering bracket is injected in the aqueous solution, in-situ solidifying is carried out, obtains in situ solid
Change tissue engineering bracket.
The present invention tests the mechanical strength of in-situ solidifying tissue engineering bracket that the present embodiment obtains in solidification 10min,
As a result as shown in Fig. 2 Fig. 2 is the mechanical strength figure of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 1～9 is obtained.
The present invention tests the porosity of the in-situ solidifying tissue engineering bracket that the present embodiment is obtained, and as a result shows, this reality
The porosity for applying the in-situ solidifying tissue engineering bracket that example is obtained is 77%.
Embodiment 7
Gelatin is prepared in 37 DEG C of deionized water the gelatin solution for obtaining that mass concentration is 25%, Ran Hou
Under 123kPa after autoclaving, bone morphogenetic protein (BMP-2) is added in gelatin solution, the concentration for obtaining BMP-2 is
The gelatin solution of 500ng/mL.
The Span-80 of 20mL atoleines and 50 μ L is added in beaker, is stirred under 37 DEG C of water bath conditions, is taken
10mL gelatin solutions are added thereto, and 10min is stirred under 200rpm rotating speeds, then keep rotating speed constant, are transferred to 4 DEG C of ice bath
Under the conditions of, 1h is persistently stirred, mixture is obtained.
Using the strainer filtering that aperture is 300 μm, by the microballoon being filtrated to get at -50 DEG C freeze-drying 24h.
With the gelatine microsphere after the quick cleaning-drying repeatedly of absolute alcohol, that is, obtain gelatine microsphere.
At 80 DEG C, 3gPLGA is dissolved in 10gNMP, adds 3gHA, stir, obtain PLGA solution.
5gPLGA solution is mixed with 5g gelatine microspheres, is stirred to homogeneous, obtain injectable in-situ solidifying organizational project
Support.Containing 9.5% PLGA, 9.5% HA, 50% gelatine microsphere in the injectable in-situ solidifying tissue engineering bracket
With 31% NMP.
Injectable in-situ solidifying tissue engineering bracket is injected in the aqueous solution, in-situ solidifying is carried out, obtains in situ solid
Change tissue engineering bracket.
The present invention tests the mechanical strength of in-situ solidifying tissue engineering bracket that the present embodiment obtains in solidification 10min,
As a result as shown in Fig. 2 Fig. 2 is the mechanical strength figure of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 1～9 is obtained.
The present invention tests the porosity of the in-situ solidifying tissue engineering bracket that the present embodiment is obtained, and as a result shows, this reality
The porosity for applying the in-situ solidifying tissue engineering bracket that example is obtained is 85%.
Embodiment 8
Gelatin is prepared in 37 DEG C of deionized water the gelatin solution for obtaining that mass concentration is 25%, Ran Hou
Under 123kPa after autoclaving, bone morphogenetic protein (BMP-2) is added in gelatin solution, the concentration for obtaining BMP-2 is
The gelatin solution of 500ng/mL.
The Span-80 of 20mL atoleines and 50 μ L is added in beaker, is stirred under 37 DEG C of water bath conditions, is taken
10mL gelatin solutions are added thereto, and 10min is stirred under 200rpm rotating speeds, then keep rotating speed constant, are transferred to 4 DEG C of ice bath
Under the conditions of, 1h is persistently stirred, mixture is obtained.
Using the strainer filtering that aperture is 300 μm, by the microballoon being filtrated to get at -50 DEG C freeze-drying 24h.
With the gelatine microsphere after the quick cleaning-drying repeatedly of absolute alcohol, that is, obtain gelatine microsphere.
At 80 DEG C, 3gPLGA is dissolved in 10gNMP, adds 3gHA, stir, obtain PLGA solution.
6gPLGA solution is mixed with 4g gelatine microspheres, is stirred to homogeneous, obtain injectable in-situ solidifying organizational project
Support.In the injectable in-situ solidifying tissue engineering bracket containing 11% PLGA, 11% HA, 40% gelatine microsphere and
38% NMP.
Injectable in-situ solidifying tissue engineering bracket is injected in the aqueous solution, in-situ solidifying is carried out, obtains in situ solid
Change tissue engineering bracket.
The present invention tests the mechanical strength of in-situ solidifying tissue engineering bracket that the present embodiment obtains in solidification 10min,
As a result as shown in Fig. 2 Fig. 2 is the mechanical strength figure of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 1～9 is obtained.
The present invention tests the porosity of the in-situ solidifying tissue engineering bracket that the present embodiment is obtained, and as a result shows, this reality
The porosity for applying the in-situ solidifying tissue engineering bracket that example is obtained is 79%.
Embodiment 9
Gelatin is prepared in 37 DEG C of deionized water the gelatin solution for obtaining that mass concentration is 25%, Ran Hou
Under 123kPa after autoclaving, bone morphogenetic protein (BMP-2) is added in gelatin solution, the concentration for obtaining BMP-2 is
The gelatin solution of 500ng/mL.
The Span-80 of 20mL atoleines and 50 μ L is added in beaker, is stirred under 37 DEG C of water bath conditions, is taken
10mL gelatin solutions are added thereto, and 10min is stirred under 200rpm rotating speeds, then keep rotating speed constant, are transferred to 4 DEG C of ice bath
Under the conditions of, 1h is persistently stirred, mixture is obtained.
Using the strainer filtering that aperture is 300 μm, by the microballoon being filtrated to get at -50 DEG C freeze-drying 24h.
With the gelatine microsphere after the quick cleaning-drying repeatedly of absolute alcohol, that is, obtain gelatine microsphere.
At 80 DEG C, 3gPLGA is dissolved in 10gNMP, adds 3gHA, stir, obtain PLGA solution.
7gPLGA solution is mixed with 3g gelatine microspheres, is stirred to homogeneous, obtain injectable in-situ solidifying organizational project
Support.In the injectable in-situ solidifying tissue engineering bracket containing 13% PLGA, 13% HA, 30% gelatine microsphere and
44% NMP.
Injectable in-situ solidifying tissue engineering bracket is injected in the aqueous solution, in-situ solidifying is carried out, obtains in situ solid
Change tissue engineering bracket.
The present invention tests the mechanical strength of in-situ solidifying tissue engineering bracket that the present embodiment obtains in solidification 10min,
As a result as shown in Fig. 2 Fig. 2 is the mechanical strength figure of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 1～9 is obtained.
In fig. 2,1 is the initial mechanical properties of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 1 is obtained；2 are
The initial mechanical properties of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 2 is obtained；3 is what the embodiment of the present invention 3 was obtained
The initial mechanical properties of in-situ solidifying tissue engineering bracket；4 is the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 4 is obtained
Initial mechanical properties；5 is the initial mechanical properties of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 5 is obtained；6 is this
The initial mechanical properties of the in-situ solidifying tissue engineering bracket that inventive embodiments 6 are obtained；7 is the original that the embodiment of the present invention 7 is obtained
The initial mechanical properties of position solidification tissue engineering bracket；8 is the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 8 is obtained
Initial mechanical properties；9 is the initial mechanical properties of the in-situ solidifying tissue engineering bracket that the embodiment of the present invention 9 is obtained.Can by Fig. 2
To find out, the present invention provide in-situ solidifying tissue engineering bracket solidification 10min in mechanical strength reach as high as 2.8MPa, greatly
Improve greatly the initial mechanical properties of support.
The present invention tests the porosity of the in-situ solidifying tissue engineering bracket that the present embodiment is obtained, and as a result shows, this reality
The porosity for applying the in-situ solidifying tissue engineering bracket that example is obtained is 71%.
As can be seen from the above embodiments, the in-situ solidifying tissue engineering bracket that the present invention is provided is with biodegradable microspheres
For pore-foaming agent, uniformly, porosity is big for the in-situ solidifying tissue engineering bracket central hole structure for obtaining, and also improves in-situ solidifying tissue
The initial mechanical properties of engineering rack, also, the preparation method of the in-situ solidifying tissue engineering bracket of present invention offer is simple, easy
Operation.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (5)
1. a kind of in-situ solidifying tissue engineering bracket, by polylactide-co-glycolide, gelatine microsphere and hydroxyapatite in 1- first
In the presence of base -2-Pyrrolidone, in-situ solidifying is formed；
In terms of mass fraction, the polylactide-co-glycolide, hydroxyapatite, gelatine microsphere and 1-Methyl-2-Pyrrolidone shape
Into solution in contain：
10% polylactide-co-glycolide, 7% hydroxyapatite, 50% gelatine microsphere and 33% 1- methyl -2- pyrroles
Alkanone；
12% polylactide-co-glycolide, 8% hydroxyapatite, 40% gelatine microsphere and 40% 1- methyl -2- pyrroles
Alkanone；
14% polylactide-co-glycolide, 9% hydroxyapatite, 30% gelatine microsphere and 47% 1- methyl -2- pyrroles
Alkanone；
9.5% polylactide-co-glycolide, 9.5% hydroxyapatite, 50% gelatine microsphere and 31% 1- methyl -2-
Pyrrolidones；
11% polylactide-co-glycolide, 11% hydroxyapatite, 40% gelatine microsphere and 38% 1- methyl -2- pyrroles
Pyrrolidone；
Or
13% polylactide-co-glycolide, 13% hydroxyapatite, 30% gelatine microsphere and 44% 1- methyl -2- pyrroles
Pyrrolidone；
The number-average molecular weight of the polylactide-co-glycolide is 50000～200000.
2. in-situ solidifying tissue engineering bracket according to claim 1, it is characterised in that the particle diameter of the gelatine microsphere is
100～450 μm.
3. in-situ solidifying tissue engineering bracket according to claim 1, it is characterised in that the in-situ solidifying organizational project
The porosity of support is 70～95%.
4. the in-situ solidifying tissue engineering bracket according to claims 1 to 3 any one, it is characterised in that the gelatin
Microballoon is the gelatine microsphere for supporting activated protein.
5. a kind of preparation method of in-situ solidifying tissue engineering bracket, comprises the following steps：
Polylactide-co-glycolide, gelatine microsphere, hydroxyapatite and 1-Methyl-2-Pyrrolidone are mixed, Jing in-situ solidifyings,
Obtain in-situ solidifying tissue engineering bracket；
In terms of mass fraction, containing 10% polylactide-co-glycolide in the solution being mixed to get, 7% hydroxyapatite,
50% gelatine microsphere and 33% 1-Methyl-2-Pyrrolidone；
12% polylactide-co-glycolide, 8% hydroxyapatite, 40% gelatine microsphere and 40% 1- methyl -2- pyrroles
Alkanone；
14% polylactide-co-glycolide, 9% hydroxyapatite, 30% gelatine microsphere and 47% 1- methyl -2- pyrroles
Alkanone；
9.5% polylactide-co-glycolide, 9.5% hydroxyapatite, 50% gelatine microsphere and 31% 1- methyl -2-
Pyrrolidones；
11% polylactide-co-glycolide, 11% hydroxyapatite, 40% gelatine microsphere and 38% 1- methyl -2- pyrroles
Pyrrolidone；
Or
13% polylactide-co-glycolide, 13% hydroxyapatite, 30% gelatine microsphere and 44% 1- methyl -2- pyrroles
Pyrrolidone；
The number-average molecular weight of the polylactide-co-glycolide is 50000～200000.
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| CN107096067A (en) * | 2017-05-03 | 2017-08-29 | 中国科学院长春应用化学研究所 | A kind of tissue engineering bracket material of fiber reinforcement in-situ solidifying pore-forming and preparation method thereof |
| CN107050506A (en) * | 2017-05-03 | 2017-08-18 | 中国科学院长春应用化学研究所 | A kind of bone tissue engineering stent material of fibre-reinforced Injectable in-situ solidification and pore-forming and preparation method thereof |
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| WO2009060473A2 (en) * | 2007-11-06 | 2009-05-14 | Panacea Biotec Limited | Injectable compositions, processes and uses thereof |
| EP2306913A4 (en) * | 2008-03-28 | 2014-06-04 | Warsaw Orthopedic Inc | Bone anchors for orthopedic applications |
| CN101507706B (en) * | 2009-03-24 | 2011-07-20 | 涂家生 | Biodegradable in-situ solidification sustained-release injector |
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