CN109276763A - Polyose modification MBG bracket, tissue recovery support and its preparation method and application - Google Patents
Polyose modification MBG bracket, tissue recovery support and its preparation method and application Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/10—Ceramics or glasses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
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- A—HUMAN NECESSITIES
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/602—Type of release, e.g. controlled, sustained, slow
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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Abstract
The present invention relates to biological medicine Material Fields, specifically, providing a kind of polyose modification MBG bracket, tissue recovery support and its preparation method and application.Polyose modification MBG bracket provided by the invention includes MBG bracket and the polysaccharide derivates that are supported on MBG bracket, MBG bracket has superior bio activity and architectural characteristic, polyose modification is carried out to MBG with polysaccharide derivates, the speed that obtained polyose modification MBG bracket can be discharged from material with the growth-delaying factor realizes the high activity sustained release of growth factor.Tissue recovery support provided by the invention and its preparation method and application has both the advantage of polyose modification MBG bracket, plays the effect of the sustained release growth factor and tissue repair.
Description
Technical field
The present invention relates to biological medicine Material Fields, in particular to a kind of polyose modification MBG bracket, tissue repair
Bracket and its preparation method and application.
Background technique
Bone tissue defect and damage have become a kind of important diseases for influencing health of people and life.It is self/different to overcome
Body bone collection bring adopts the problems such as bone limited source, second operation pain and immunological rejection and propagation of potential pathogeny, development
Artificial bone renovating material becomes research hotspot in recent years.However, it is suitable to be applied to clinical artificial bone renovating material at present
It is limited, it mainly include calcium phosphate (such as tricalcium phosphate, porous hydroxyapatite bioceramic and calcium phosphate bone cement), biological micro-
The type products such as crystal glass and collagen.Although these materials can to a certain extent inducting osseous tissue growth and new bone apposition,
But the activity that them are compared with autologous bone still is apparent not enough.
Outstanding problems, the growth factor modifieds such as, osteanagenesis speed low for clinical bone renovating material bioactivity is slow are structures
Build the effective way of high activity bone repair materials.A large number of studies show that growth factor effectively can promote specific cells to wound site
Migration, differentiation, participate in the adjusting of bone defect healing.But since growth factor itself is vulnerable to internal external condition (such as body fluid, temperature
Degree, pH environment and organic reagent etc.) influence, there are the following problems in application process: 1. half-life short, easily because dilute
It releases, be metabolized and digest and inactivate；2. potential excess toxic side effect；3. it is expensive, with the production of Medtronic Inc. of the U.S.
For rhBMP-2, rhBMP-2 price is up to 12000 yuan/milligram.Due to being influenced by vivo environment, only small part BMP-2
It is practical to play bone inductive effect, have to increase dosage (Medtronic Inc. to reach therapeutic effect when clinical use thus
RhBMP-2 content is 4mg/ piece in the Infuse of production), this does not only result in medical expense rising, more increases and is excessively used
The potential risk of BMP-2.
To solve this problem, researchers at home and abroad have developed a variety of growth factor carrying methods, comprising: physics is inhaled
The embedding of attached, microballoon, multicoat be compound, ion complexation, affinity molecule combine and covalent bonding etc..But these methods still cannot
Reach long-acting slow-release while maintaining growth factor activity well, while load efficiency is low, stability is insufficient, and wherein
Application of most of method on bone renovating material be not strong.
Therefore, it designs and prepares and had not only been able to satisfy Bone Defect Repari primary demand, but also can realize growth factor high activity loading simultaneously
Artificial bone renovating material be still a challenging job.
In view of this, the present invention is specifically proposed.
Summary of the invention
It is an object of the present invention to provide a kind of polyose modification MBG brackets, can be used as tissue engineering material, efficiently
It is growth factor-loaded, the long stable effect sustained release growth factor.
It is another object of the present invention to provide the preparation methods of polyose modification MBG bracket, and this method is simple to operation,
Can industrial operation, can efficiently, quickly, the polyose modification MBG bracket of batch output high quality.
It is another object of the present invention to provide the applications of polyose modification MBG bracket, load for growth factor optimizing, group
Design, preparation and the performance study of weaver's journey repair materials provide technical support.
It is another object of the present invention to provide a kind of tissue recovery supports, being capable of long stable effect sustained release high activity growth
The factor, effectively progress tissue repair.
It is another object of the present invention to provide the preparation methods of tissue recovery support, and this method is simple and easy to operate, raw
It produces at low cost.
A kind of polyose modification MBG bracket, the polyose modification MBG bracket include MBG bracket and are supported on MBG bracket
Polysaccharide derivates.
Further, the MBG bracket is three-dimensional porous connected network structure, has 200-500 μm of connection macropore, 0.5-
5 μm of surface micropore and aperture are the order mesoporous of 6-9nm；
Preferably, the skeleton hole wall thickness of the MBG bracket is 40-100 μm.
Further, amino is modified on the MBG bracket.
Further, the polysaccharide derivates include sulfated chitosan, hyaluronic acid, chondroitin sulfate, acetyl sulfate liver
At least one of element or heparin, preferably sulfated chitosan；
The polysaccharide derivates quality be MBG bracket quality 0.001%-0.3%, preferably 0.003%.
A kind of preparation method of above-mentioned polyose modification MBG bracket, polysaccharide derivates are carried on MBG bracket, are obtained more
Sugar-modified MBG bracket.
Further, by polysaccharide derivates negative-pressure adsorption on MBG bracket, freeze-drying obtains polyose modification MBG bracket.
Further, further include that MBG bracket is first subjected to amination moditied processing, be then again carried on polysaccharide derivates
On MBG bracket, the step of obtaining polyose modification MBG bracket；
Preferably, the step of amination moditied processing includes: by MBG bracket, low-carbon alcohols and amino silicane coupling agent
24-96h is mixed, obtains amido modified MBG bracket, wherein MBG bracket quality g, low-carbon alcohols volume ml and amino silicane coupling agent
The ratio of volume ml is 1:150-250:3-8；
Preferably, the amino silicane coupling agent includes gamma-aminopropyl-triethoxy-silane, γ-aminopropyl trimethoxy
Silane or N- β (aminoethyl)-γ-aminopropyltrimethoxysilane, preferably gamma-aminopropyl-triethoxy-silane；
Preferably, the low-carbon alcohols include methanol, ethyl alcohol, propyl alcohol, isopropanol, butanol, isobutanol, preferably ethyl alcohol, into
One step is preferably dehydrated alcohol；
Preferably, the method also includes by after MBG bracket, low-carbon alcohols and amino silicane coupling agent mixing 24-96h again into
The step of row washing；
Preferably, the step of washing includes: independently to be washed 3 times with ethyl alcohol and ultrapure water, obtains ammonia after dry
Base modifies MBG bracket；
Preferably, by slurry cast to template, removing template is removed after dry, obtains the MBG bracket, wherein the slurry
Material includes the mixing of MBG microballoon, micropore pore-forming reagent and MBG colloidal sol, the quality of MBG microballoon, micropore pore-forming reagent and MBG colloidal sol
Than for 0.5-1:0.25-1:1-7；
Preferably, the stock quality g and template volume cm3Ratio are as follows: 0.3-0.6:1；
Preferably, the micropore pore-forming reagent includes polyacrylic acid microballoon, polystyrene microsphere, polymethyl methacrylate
Microballoon；
Preferably, the partial size of the polyacrylic acid microballoon is 5-30 μm；
Preferably, the viscosity of the MBG colloidal sol is 4-6 × 104mPa·s；
Preferably, the partial size of the MBG microballoon is 8-12 μm；
Preferably, the MBG microballoon is dry by MBG colloidal sol and ball milling sieving is prepared；
Preferably, the template is polyurethane sponge or polyvinylalcohol sponge；
Preferably, for the template by pretreatment, the pretreatment includes: to clean 1.5-2.5h with NaOH solution, wherein
The mass fraction of NaOH solution is 10-20%；
Preferably, removing template is gone by the method for sintering, the sintering temperature is 550-650 DEG C, sintering time 5-7h.
The polyose modification MBG bracket that the present invention provides above-mentioned polyose modification MBG bracket again or preparation method obtains is as follows
A)-D) application in any one:
A) growth factor loads；
B) growth factor slow-release；
C) growth factor activity regulates and controls；
D) tissue renovation material and its preparation.
A kind of tissue recovery support, the polyose modification being prepared including above-mentioned polyose modification MBG bracket or preparation method
MBG bracket and the growth factor for being carried on polyose modification MBG bracket；
Preferably, the growth factor includes at least one of BMP-2, VEGF or FGF, preferably BMP-2；
The growth factor quality be polyose modification MBG bracket quality 0.001%-0.1%, preferably 0.0015%.
A kind of preparation method of above-mentioned tissue recovery support, will it is raw in factor negative-pressure adsorption on polyose modification MBG bracket,
Freeze-drying obtains tissue recovery support.
Compared with prior art, the invention has the benefit that
Polyose modification MBG bracket provided by the invention, raw material include the polysaccharide derivates of MBG bracket and modification MBG bracket.
MBG bracket has superior bio activity and architectural characteristic affine based on polysaccharide derivates and growth factor heparin binding domain simultaneously
This characteristic of property, carries out polyose modification to MBG bracket with polysaccharide derivates, obtains polyose modification MBG bracket.The polyose modification
For MBG bracket of the MBG bracket compared to no any modification, growth factor-loaded load capacity is higher, while growth factor
Stability it is also more preferable.The speed that polyose modification MBG bracket can be discharged from material with the growth-delaying factor, realize growth because
The high activity sustained release of son.Polyose modification MBG bracket can be used as tissue engineering material, have good biocompatibility, Neng Gougao
The advantages of imitating the growth factor-loaded and long stable effect sustained release growth factor, and due to can block growth factor and growth because
The combination of sub- antagonist, to improve growth factor activity in vivo.
The present invention provides a kind of preparation method of polyose modification MBG bracket, and the preparation method is simple and easy to operate, can industrialize
Operation, so as to efficient, quick, batch output polyose modification MBG bracket.
Tissue recovery support provided by the invention, the advantage with above-mentioned polyose modification MBG bracket, it is thus possible to stablize length
Effect sustained release high activity growth factor, effective reparation that must carry out tissue defect and damage.
Detailed description of the invention
Fig. 1 is that sol-gal process prepares mesoporous activated glass flow chart in the embodiment of the present invention 1；
Fig. 2 is the preparation step flow chart of MBG bracket in the embodiment of the present invention 2；
Fig. 3 is the amination preparation step flow chart of MBG bracket in the embodiment of the present invention 3；
Fig. 4 is the synthesis step flow chart of 26SCS in the embodiment of the present invention 4；
Fig. 5 is MBG-NH in the embodiment of the present invention 52The preparation flow figure of/BMP-2/26SCS compound rest；
Fig. 6 A is in test example 1 of the present invention to the amination MBG/NH prepared in embodiment 32Bracket carries out infrared spectroscopy inspection
Survey result figure；
Fig. 6 B is in test example 1 of the present invention to the amination MBG/NH prepared in embodiment 32Bracket carries out ninhydrin detection
Result figure；
Fig. 6 C is to scheme (200-500 μm) to the SEM that MBG bracket pattern in embodiment 3 carries out in test example 1 of the present invention；
Fig. 6 D is to scheme (0.5-5 μm) to the SEM that MBG bracket pattern in embodiment 3 carries out in test example 1 of the present invention；
Fig. 6 E is to scheme (7.5nm) to the TEM that MBG bracket pattern in embodiment 3 carries out in test example 1 of the present invention；
Fig. 7 A is to carry out infrared spectroscopy testing result figure to the 26SCS prepared in embodiment 4 in test example 2 of the present invention；
Fig. 7 B is to carry out toluidine blue testing result figure to the 26SCS prepared in embodiment 4 in test example 2 of the present invention；
Fig. 8 is the release in vitro behavioral value result figure of BMP-2 in test example 3 of the present invention；
Fig. 9 is BMSCs cell in test example 4 of the present invention in MBG, MBG/BMP-2 and MBG-NH2/ BMP-2/26SCS bracket
On proliferative conditions statistical chart；
Figure 10 A is cell ALP vigor (3d, 7d) measurement result figure in test example 5 of the present invention；
Figure 10 B is that cell ALP dyes (3d) measurement result figure in test example 5 of the present invention.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.
A kind of polyose modification MBG bracket, raw material include the polysaccharide derivates of MBG bracket and modification MBG bracket.
MBG (mesoporous bioactive glass, mesoporous activated glass) is living for novel ordered nano mesoporous biological
Property glass.Sodium is free of in component, Si, Ca molar ratio are 80:15；With uniform nanoscale meso-hole structure and higher compare table
Area.MBG bioactivity with higher, good degradability, with excellent biomineralization activity in body fluid, simultaneously
With good biocompatibility.
Growth factor, which has, significantly promotes specific cells to wound site migration, differentiation, and participates in tissue defect healing
Effect.Due to there are heparin binding domain, being combined repeatedly with growth factor by being modified on MBG bracket inside growth factor and
The heparin of interaction, can the speed that is discharged from MBG bracket of the growth-delaying factor, and by regulation ligand to its cell membrane institute
The receptor signal of expression improves the bioactivity of growth factor.Meanwhile the polysaccharide with heparin characteristic can also be used to modify
MBG bracket.
MBG bracket has superior bio activity and architectural characteristic, while based on polysaccharide derivates and growth factor heparin knot
This characteristic of the compatibility in domain is closed, polyose modification is carried out to MBG bracket with polysaccharide derivates, obtains polyose modification MBG bracket.It should
For MBG bracket of the polyose modification MBG bracket compared to no any modification, growth factor-loaded load capacity is higher, simultaneously
The stability of growth factor is also more preferable.The speed that polyose modification MBG bracket can be discharged from material with the growth-delaying factor, it is real
The high activity sustained release of existing growth factor.Polyose modification MBG bracket can be used as tissue engineering material, have biocompatibility
It is good, can high-efficient carrier growth factor and the advantages of the long stable effect sustained release growth factor, and due to can block growth because
The combination of son and growth factor antagonist, to improve growth factor activity in vivo.
In the present invention, one is preferably carried out in mode, and MBG bracket is three-dimensional porous connected network structure, has 200-
500 μm of connection macropores, 0.5-5 μm of surface micropore and aperture are the order mesoporous of 6-9nm.In the structure of the MBG bracket,
The porosity of MBG bracket can achieve 85% or more, and according to the hole of the different adjustable MBG brackets of application scenarios
Rate, interconnected macropore are conducive to histiocytic growth, connection macropore, surface micropore and it is order mesoporous be MBG bracket not
With the structure and morphology under amplification factor, this structure and morphology of MBG bracket determines that MBG bracket has very high specific surface area.
It, can be by carrying out amination processing to MBG rack surface in addition, on the basis of MBG bracket high-specific surface area
For one step using the strong electrostatic interaction between amino and polysaccharide derivates, the common height for realizing polysaccharide derivates on MBG bracket is negative
It carries.Entire rack surface is all modified with polysaccharide derivates, and due to the affine combination of growth factor and polysaccharide derivates, growth
The factor can with steady load in the connection macropore of MBG bracket, surface micropore and it is order mesoporous in, and then realize growth factor length
Effect release and bioactivity maintain.Since polyose modification MBG bracket can discharge growth factor with long-acting stabilization, and due to connection
Macropore is conducive to nutriment transmission and tissue cell growth, effectively facilitates so tissue repair is available.
In some embodiments of the present invention, the skeleton hole wall thickness of MBG bracket is 40-100 μm.The skeleton hole of MBG bracket
Wall is excessively thin, and the overall stability of MBG bracket is poor, is unfavorable for the holding of MBG supporting structure；The skeleton hole wall of MBG bracket is blocked up,
The connection macro porosity led of MBG bracket will become smaller, and the blood vessel and cambium being unfavorable in tissue regeneration processes are grown into.MBG
When the skeleton pore wall thickness of bracket is 40-100 μm, have MBG bracket
There is high porosity, is conducive to growing into for tissue.It is 40 μm that the skeleton pore wall thickness of MBG bracket is typical but non-limiting, 45 μ
M, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm or 100 μm.
In the present invention, one is preferably carried out in mode, is modified with amino on MBG bracket.Amino is carried out to MBG bracket to repair
Decorations, by electrostatic interaction, elecrtonegativity sulfonic acid group and amino stable bond in polysaccharide derivates further improve polysaccharide and spread out
The load stability of biology.Amino is first modified on MBG bracket, by the stable bond of polysaccharide derivates and amino, further
Ensure the steady load of polysaccharide derivates, and then improves the sustained release and activity of growth factor, so that polyose modification MBG bracket
Stability further enhances.
In certain embodiments of the present invention, polysaccharide derivates include sulfated chitosan, hyaluronic acid, chondroitin sulfate
At least one of element, Heparan sulfate or heparin.
In the present invention, one is preferably carried out in mode, and polysaccharide derivates are sulfated chitosan.Sulfated chitosan (2-N, 6-
O-sulfated chitosan, 26SCS) safety is good, and economic and easy large-scale production has no toxic side effect to body, and send out
Bright people has found that sulfated chitosan has good effect on regulation growth factor activity and release by Experimental Comparison.
In the present invention, one is preferably carried out in mode, and polysaccharide derivates quality is the 0.001%- of MBG bracket quality
0.3%, preferably 0.003%.Polysaccharide derivates quality is very few and excessive, all will affect the synergistic effect with growth factor, drop
The bioactivity of low polyose modification MBG bracket；The typical but non-limiting quality of polysaccharide derivates is MBG bracket quality
0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2% or 0.3%.
The present invention provides the preparation method of above-mentioned polyose modification MBG bracket, and polysaccharide derivates are carried on MBG bracket,
Obtain polyose modification MBG bracket.The preparation method is simple and easy to operate, can industrial operation, so as to it is efficient, quick, batch
Output polyose modification MBG bracket.
In certain embodiments of the present invention, the saturation adsorption volume for measuring MBG bracket, will require the polysaccharide of content to spread out
Biological solution fixing fabric structure is the saturation adsorption volume of bracket, and for negative-pressure adsorption on MBG bracket, freeze-drying obtains polyose modification MBG
Bracket.It will be added dropwise with the polysaccharide solution of bracket saturated volume in the uniform load for being conducive to polysaccharide derivates on MBG bracket, lead to
It crosses freeze-drying polysaccharide derivates are stable on MBG bracket, and the destruction of polysaccharide derivates molecular structure will not be caused, to realize
Effective high activity loading of polysaccharide derivates, is conducive to the combination of subsequent polysaccharide derivates and growth factor.Due to growth factor
It is uniformly largely stable to be carried on polyose modification MBG bracket, so good sustained release and activity dimension may be implemented in growth factor
It holds.
In the present invention, one is preferably carried out in mode, further includes that MBG bracket is first carried out amination moditied processing, then
The step of polysaccharide derivates are carried on MBG bracket again, obtain polyose modification MBG bracket.First amino is carried out to MBG bracket to repair
Decorations, then by amino stable bond polysaccharide derivates, obtain the polyose modification MBG bracket of high quality.
In the present invention, one is preferably carried out in mode, by MBG bracket, low-carbon alcohols and amino silicane coupling agent mixing 24-
96h obtains amido modified MBG bracket, wherein MBG bracket quality g, low-carbon alcohols volume ml and amino silicane coupling agent volume ml
Ratio be 1:150-250:3-8.In low-carbon alcohols solvent environment, the amino in amino silicane coupling agent is transformed into MBG bracket
On, realize amido modified MBG bracket.The ratio of MBG bracket quality g, low-carbon alcohols volume ml and amino silicane coupling agent volume ml
Typical but non-limiting is 1:150:3,1:250:3,1:150:8,1:250:8 or 1:200:6.Amino silicane coupling agent mistake
It is more, it will lead to the increase of production cost；Amino silicane coupling agent is very few, and MBG bracket is amido modified few, the load of polysaccharide derivates
Stability reduces.
The present invention one be preferably carried out in mode, amino silicane coupling agent include gamma-aminopropyl-triethoxy-silane,
γ-aminopropyltrimethoxysilane or N- β (aminoethyl)-γ-aminopropyltrimethoxysilane, preferably three second of γ-aminopropyl
Oxysilane.
In the present invention, one is preferably carried out in mode, and low-carbon alcohols include methanol, ethyl alcohol, propyl alcohol, isopropanol, butanol, different
Butanol, preferably ethyl alcohol, further preferably dehydrated alcohol.
It further include by MBG bracket, low-carbon alcohols and amino silicane coupling agent mixing 24- in some embodiments of the present invention
The step of being washed again after 96h.Impurity and unreacted amino silicone can be removed by washing to amido modified MBG bracket
The solution such as alkane coupling agent are conducive to the polysaccharide derivates load in later period.
The step of one is preferably carried out in mode in the present invention, washing includes: independently to be washed with ethyl alcohol and ultrapure water
It washs 3 times, obtains amido modified MBG bracket after dry.
In some embodiments of the present invention, by slurry cast to template, removing template is removed after dry, obtains MBG bracket,
Wherein, slurry includes the mixing of MBG microballoon, micropore pore-forming reagent and MBG colloidal sol, and MBG microballoon, micropore pore-forming reagent and MBG are molten
The mass ratio of glue is 0.5-1:0.25-1:1-7.It is simple to operation using this method, MBG bracket is prepared and does not have brokenly completely
Damage.Allow stabilization of the slurry in sponge template by the rational proportion of MBG microballoon, micropore pore-forming reagent and MBG colloidal sol
One-pass molding, and obtain the MBG bracket of stable structure, high mechanical strength.In the present invention, MBG microballoon, micropore pore-forming reagent and
The typical but non-limiting mass ratio of MBG colloidal sol is 0.5:0.5:7,0.75:0.75:7,1:1:7,0.5:0.5:6,0.75:
0.75:6 or 1:1:6.
In the present invention, one is preferably carried out in mode, stock quality g and template volume cm3Ratio are as follows: 0.3-0.6:
1.Slurry is very few, and slurry is not filled up completely in template, causes the MBG rack mechanical strength of preparation low, cannot reach expected
Structure composition；Slurry is excessive, and macropore connectivity and porosity decline are unfavorable for growing into for tissue and produce with nutriment/and metabolism
The exchange of object.Stock quality g and template volume cm3Ratio when being 0.3-0.6:1, it is good to obtain mechanical strength, structure shape
The uniform MBG bracket of looks.In the present invention, stock quality g and template volume cm3Ratio it is typical but non-limiting be 0.3:
1,0.4:1,0.5:1,0.55:1 or 0.6:1, every 1cm3Template be preferably poured 0.55g slurry.
In certain embodiments of the present invention, micropore pore-forming reagent includes polyacrylic acid microballoon, polystyrene microsphere, gathers
Methyl methacrylate microballoon.Micropore pore former is the surface micropore in order to prepare MBG bracket, these micropores are conducive to cell
Adherency, biomineralization and Integrated implant.
In certain embodiments of the present invention, the partial size of polyacrylic acid microballoon is 5-30 μm.The grain of polyacrylic acid microballoon
Typical but non-limiting diameter is 5 μm, 10 μm, 15 μm, 20 μm, 25 μm or 30 μm.
In certain embodiments of the present invention, the viscosity of MBG colloidal sol is 4-6 × 104mPa·s.Inventor passes through test
It was found that slurry prepared by the MBG colloidal sol of the viscosity just can guarantee the steady load and molding in MBG slurry in sponge template.
In certain embodiments of the present invention, the partial size of MBG microballoon is 8-12 μm.The partial size of MBG microballoon will affect system
The mechanical strength of standby MBG bracket, MBG microballoon it is oversized or too small, will all be unfavorable for mechanics of materials stability.
In certain embodiments of the present invention, MBG microballoon is prepared by the drying of MBG colloidal sol and ball milling sieving.
In certain embodiments of the present invention, template is polyurethane sponge or polyvinylalcohol sponge.Polyurethane sponge and
Polyvinylalcohol sponge belongs to low-density, and high-specific surface area and uniform, the controllable material of structure can be used as macropore template
Agent.
In the present invention, one is preferably carried out in mode, and template is by pretreatment: 1.5-2.5h is cleaned with NaOH solution,
In, the mass fraction of NaOH solution is 10-20%.Pretreated purpose is thoroughly to be cleaned template, improves MBG slurry
The load of material.
In the present invention, one is preferably carried out in mode, removes removing template, sintering temperature 550-650 by the method for sintering
DEG C, sintering time 5-7h.Template not only can remove using the sintering method of this condition, can also protect to the full extent
The integrality of MBG internal stent meso-hole structure, so that the meso-hole structure of MBG bracket will not collapse because of high temperature.Sintering temperature mistake
The low or time is very few, and template removal not exclusively, is easy to produce template residual；Sintering temperature is excessively high or overlong time, easily causes MBG
The destruction of bracket meso-hole structure.Sintering temperature is typical but be without limitation 550 DEG C, 560 DEG C, 570 DEG C, 580 DEG C, 590 DEG C,
600 DEG C, 610 DEG C, 620 DEG C, 630 DEG C, 640 DEG C or 650 DEG C；Sintering time is typical but is without limitation 5h, 6h or 7h.
In the present invention, one is preferably carried out in mode, the preparation of MBG bracket the following steps are included:
(a) use polyurethane sponge as macropore template.After polyurethane sponge is processed into dimension, in 20%NaOH
It is impregnated in solution and rubs pretreatment 2h；
(b) by the MBG colloidal sol prepared revolving instrument on 60 DEG C of revolving 30min, obtain viscosity be 4 × 104-6×
104The viscous sol of mPas；
(c) it is sieved the drying of sol-fraction prepared by (b) and ball milling to obtain the MBG microballoon that partial size is about 10 μm；
(d) by MBG microballoon prepared by (c) and micropore pore-forming reagent (polyacrylic acid microballoon, 20 μm), with 1:1:6, (MBG is micro-
Ball: polyacrylic acid microballoon: MBG colloidal sol) mass ratio be uniformly mixed into (b) revolving treated MBG colloidal sol, and with 0.55g/
cm3Stock quality/sponge volume ratio be cast on polyurethane sponge；
(e) 600 DEG C of high temperature sintering 6h (1 DEG C/min of heating rate) go template after 60 DEG C of constant temperature convection oven drying, finally
Obtain the macropore/micropore/mesopore bioactive glass bracket with certain mechanical strength.
The preparation method process flow of above-mentioned MBG bracket is simple, can be operated with heavy industrialization, by polyurethane sea
The adjustment of continuous template obtains the MBG bracket of different contour structures, to meet different actual needs.The MBG branch that this method obtains
Frame structure is uniform, and microscopic appearance is stablized, and is conducive to the amino in later period and/or the operation of polyose modification technique.
In the present invention, one is preferably carried out in mode, can pass through 3D according to the actual needs and requirement of tissue repair
The mode of printing prepares the template of needs, so that the MBG bracket of needs be prepared, or directlys adopt the mode system of 3D printing
It is standby to obtain MBG bracket.
In the present invention, one is preferably carried out in mode, the preparation of MBG colloidal sol the following steps are included:
(a) 30 DEG C of water-baths of reaction vessel are added in 300mL dehydrated alcohol and 6g 0.5M HCl；
(b) 24g F127 (EO is added106PO70EO106) be vigorously stirred to being completely dissolved, solution is in clear state；
(c) 6.56g four water-calcium nitrate (Ca (NO is added3)2·4H2O), stirring 10min is completely dissolved to it；
(d) 1.38g triethyl phosphate (TEP) is added；
(e) 31.2g ethyl orthosilicate (TEOS) is added dropwise；
(f) 30 DEG C of water-baths are kept to be vigorously stirred reaction for 24 hours after reagent is added.
This method simple process, the MBG sol ingredient being prepared are stablized.
The present invention one be preferably carried out in mode, MBG bracket it is amidized preparation the following steps are included:
(a) 1g MBG bracket is weighed, 200mL dehydrated alcohol is added；
(b) 6mL (3- aminopropyl) triethoxysilane (APTES) is added dropwise under oxygen free condition dropwise；
(c) it is protected from light and is stirred to react 72h；
(d) to be washed respectively 3 times with dehydrated alcohol, ultrapure water after reaction；
(e) 60 DEG C of drying obtain amido modified MBG bracket overnight.
The amidized preparation method of MBG bracket is simple and easy to operate, is suitble to industrialized production.
The polyose modification MBG bracket that the present invention provides above-mentioned polyose modification MBG bracket or preparation method is prepared is in A)-
D) the application in any one:
A) growth factor loads；
B) growth factor slow-release；
C) growth factor activity regulates and controls；
D) tissue renovation material and its preparation.
It is matrix based on MBG provided by the invention, carries out the polysaccharide that polyose modification or amino and polysaccharide are modified jointly
MBG bracket is modified, there is stable, high load amount binding growth factor advantage, while improving the activity of growth factor, and
The had good sustained release effect of growth factor.Application using the field of biomedical materials of this mechanism and effect can be the present invention
The polyose modification MBG bracket of offer or the application range of bone repairing support.The application range of tissue renovation material is typical but non-limit
Property processed be include bone defect healing, periodontal reparation, skin repair etc..
A kind of tissue recovery support, the polysaccharide being prepared including above-mentioned polyose modification MBG bracket or above-mentioned preparation method
Modification MBG bracket and the growth factor for being carried on polyose modification MBG bracket.Tissue recovery support provided by the invention has upper
State the advantage of polyose modification MBG bracket, it is thus possible to which long stable effect is sustained high activity growth factor, effective to carry out tissue defect
With the reparation of damage.
In the present invention, one is preferably carried out in mode, and growth factor includes at least one of BMP-2, VEGF or FGF,
Preferably BMP-2.Growth factor significantly promotes specific cells to wound site migration, differentiation due to having, and participates in bone defect
The effect of healing is to generally acknowledge the effective way for improving material activity.Wherein, BMP-2 (bone morphogenetic protein
2, bone morphogenetic protein 2) it is that strongest growth factor is acted on to bon e formation in known bioactie agent, it is filled between capable of inducing
Matter stem cell is to cartilage cell and osteoblast directed differentiation, hence it is evident that promotes skeletonization and shortens bone tissue healing time.
In the present invention, one is preferably carried out in mode, and growth factor quality is the 0.001%- of polyose modification MBG mass
0.1%, preferably 0.0015%.It can be adjusted according to experiment demand, factor content is very few to be unfavorable for regeneration, excessively
It can cause inflammation, and cost is too high.
The present invention provides a kind of preparation method of above-mentioned bone repairing support, by growth factor negative-pressure adsorption in polyose modification
On MBG bracket, freeze-drying obtains tissue recovery support.This method is fast and convenient, be conducive to by the way of negative-pressure adsorption growth because
Son is rapidly and uniformly carried on polyose modification MBG rack surface.This method is suitable for batch production.
In order to help to further understand the present invention, technical solution of the present invention is carried out now in conjunction with preferred embodiment detailed
Explanation.
Unless otherwise specified, instrument or reagent commercially obtain in the embodiment of the present invention.The present invention is real
It applies test method used in example unless otherwise specified, is conventional method.
The preparation of 1 mesoporous activated glass (MBG) of embodiment
The preparation step flow chart of mesoporous activated glass (MBG) is as shown in Figure 1.Specific step is as follows:
(a) 30 DEG C of water-baths of reaction vessel are added in 300mL dehydrated alcohol and 6g 0.5M HCl；
(b) 24g F127 (EO is added106PO70EO106) be vigorously stirred to being completely dissolved, solution is in clear state；
(c) 6.56g four water-calcium nitrate (Ca (NO is added3)2·4H2O), stirring 10min is completely dissolved to it；
(d) 1.38g triethyl phosphate (TEP) is added；
(e) 31.2g ethyl orthosilicate (TEOS) is added dropwise；
(f) 30 DEG C of water-baths are kept to be vigorously stirred reaction for 24 hours after reagent is added.
The preparation of 2 MBG bracket of embodiment
The preparation step flow chart of MBG bracket is as shown in Figure 2.Specific step is as follows:
(a) use polyurethane sponge as macropore template.After polyurethane sponge is processed into dimension, in 20%NaOH
It is impregnated in solution and rubs pretreatment 2h；
(b) by the MBG colloidal sol prepared revolving instrument on 60 DEG C of revolving 30min, obtain viscosity be 4 × 104-6×
104The viscous sol of mPas；
(c) it is sieved the drying of sol-fraction prepared by (b), ball milling to obtain the MBG microballoon that partial size is about 10 μm；
(d) by MBG microballoon prepared by (c) and micropore pore-forming reagent (polyacrylic acid microballoon, 20 μm), with 1:1:6, (MBG is micro-
Ball: polyacrylic acid microballoon: MBG colloidal sol) mass ratio be uniformly mixed into (b) revolving treated MBG colloidal sol, and with 0.55g/
cm3Stock quality/sponge volume ratio be cast on the polyurethane sponge of well cutting；
(e) 600 DEG C of high temperature sintering 6h (1 DEG C/min of heating rate) go template after 60 DEG C of constant temperature convection oven drying, finally
Obtain the macropore/micropore/mesopore bioactive glass bracket with certain mechanical strength.
The amination of 3 MBG bracket of embodiment
The amination preparation step flow chart of MBG bracket is as shown in Figure 3.Specific step is as follows:
(a) the MBG bracket prepared in 1g embodiment 2 is weighed, 200mL dehydrated alcohol is added；
(b) 6mL (3- aminopropyl) triethoxysilane (APTES) is added dropwise under oxygen free condition dropwise；
(c) it is protected from light and is stirred to react 72h；
(d) to be washed respectively 3 times with dehydrated alcohol, ultrapure water after reaction；
(e) 60 DEG C of drying obtain amido modified MBG bracket overnight.
4 2-N of embodiment, the synthesis of 6-O- sulfated chitosan (26SCS)
The synthesis step flow chart of 26SCS is as shown in Figure 4.Specific step is as follows:
(a) prepared by sulfonated reagent: measuring 50mL n,N-Dimethylformamide (DMF) liquid, is added to tri- mouthfuls of 500mL burnings
In bottle, ice bath is cooled to 0-4 DEG C；5mLHClSO is added dropwise under oxygen free condition dropwise3, magnetic agitation reaction mixture (DMFSO3)
And cooling reaches room temperature.
(b) prepared by sulfated chitosan: accurately weighing 2.5g chitosan into 500mL three-necked flask, 50mL formamide is added
With 2mL formic acid, mechanical stirring dissolves chitosan sufficiently；At 50 DEG C of oxygen free condition, step (1) is slowly added dropwise into three-necked flask
The sulfonated reagent of preparation is vigorously stirred reaction 2h.
(c) it post-processes: to after the reaction was completed, pour into 500mL dehydrated alcohol, obtaining precipitated product；Vacuum filtration, anhydrous second
Alcohol is dissolved in ultrapure water after washing 3 times；System pH is adjusted to 7.4 with 1M HCl or 1M NaOH；It is centrifuged 15min, takes supernatant
The 3d that dialyses is transferred in the bag filter that molecular cut off is 14KDa；Final product is obtained after freeze-drying.
The modification of the loading and 26SCS of 5 growth factor of embodiment
BMP-2 and 26SCS are loaded using negative-pressure adsorption-desivac, flow chart of steps is as shown in Figure 5.It is specific
Operating procedure is as follows:
(a) it in order to guarantee that various brackets load quantitative BMP-2 and 26SCS, and is evenly distributed in bracket, first to each
The saturation adsorption volume of bracket is measured；
(b) using the BMP-2 of the PBS buffer solution dissolution fixed amount of corresponding saturation adsorption volume；
(c) prepared BMP-2 solution is added dropwise on corresponding sterilizing bracket using liquid-transfering gun；
(d) 4 DEG C of negative-pressure adsorptions and the load of 26SCS is carried out after being lyophilized, the same BMP-2 of the mode of loading of 26SCS finally obtains
MBG-NH2/ BMP-2/26SCS or MBG/BMP-2/26SCS.
Test example 1
The for the benefit of load of sulfated chitosan 26SCS utilizes rack surface ammonia after carrying out amination processing to MBG bracket
The strong electrostatic interaction of base and 26SCS sulfonate radical is combined.It can be seen that material is in 3463 Hes from infrared spectrum analysis
3228cm-1There is-NH at place2The N-H stretching vibration peak (Fig. 6 A) of group.(such as using ammonia, ammonium salt and compound containing free amine group
Primary amine etc.) and producible this characteristic of bluish violet condensation product Ruhemann's Purple of ninhydrin heat together, apparent sieve of rack surface can be observed
Graceful purple colour developing (Fig. 6 B).
Ninhydrin detection method is as follows: weighing 0.64g ninhydrin and is dissolved in the mixed of 4mL monosodium citrate (0.5M) and glycerine
It closes in solution, wherein monosodium citrate solution and glycerine volume ratio are 1:2；The bracket of amination processing is transferred to ninhydrin
37 DEG C of incubation 15min in solution, carry out observation of taking pictures to sample using digital camera.As a result as shown in Figure 6B.
Above-mentioned experiment confirms that rack surface has new amino to introduce, MBG-NH2Bracket is successfully prepared.
Meanwhile the bracket pattern that amination is handled is observed by scanning electron microscope and transmission electron microscope, the results showed that should
There is bracket 200-500 μm high macropore, 0.5-5 μm of abundant surface micropore and the aperture of being connected to be situated between for the high-specific surface area of 7.5nm
Hole (Fig. 6 C-6E).
From SEM picture (bracket can be obviously observed in Fig. 6 C and 6D) and is connected to macroporous network by 200-500 μm high and is formed, greatly
Pore morphology is uniform, complete, about 40-60 μm of skeleton hole wall thickness, and does not observe tomography and non-continuous event in figure, and bracket
Surface is evenly distributed with the micropore that diameter is 2 μm.To be loaded suitable for BMP-2, prepare aperture and BMP-2 size (3 × 3.5 ×
7nm3) comparable meso-hole structure.As shown in TEM photo (Fig. 6 E), aperture is that the regular ordered nano duct of 7.5nm is distributed in branch
Inside frame, it is theoretically suitable for the loading of BMP-2.
Above-mentioned experiment confirms that the activity glass bracket with hierarchical porous structure is successfully prepared, and amination does not destroy
The multilevel structure characteristic of bracket.
Test example 2
26SCS prepared by embodiment 4 carries out infrared analysis, and Fig. 7 A is the infared spectrum of 26SCS, poly- compared to original shell
Sugar, sulfated chitosan is respectively in 1222cm-1、806cm-1There is the characteristic peak of S=O and C-O-S.Further, amination is changed
MBG-NH after property2Bracket is compound with 26SCS, by Toluidine blue staining, to the sulfated chitosan distribution situation of rack surface
It is observed.Its dyeing theory is that the monomer (the aobvious blue of monomer) of dyestuff in toluidine blue solution is complexed with polyanionic materials
Afterwards, dye molecule can be grouped to polymer displaing amaranth.Since 26SCS is a kind of polyanionic polysaccharide with sulfonate radical, because
This can qualitative observation MBG-NH by Toluidine blue staining method2The 26SCS loading condition of rack surface.
Toluidine blue detection method is as follows: weighing 0.5mg toluidine blue and 20mg NaCl is added to 10mL 0.01M HCl
In, the bracket for being compounded with 26SCS is transferred in above-mentioned toluidine blue solution and is incubated at room temperature 6h, using digital camera to sample into
Capable observation of taking pictures.Can be observed from Fig. 7 B colored graph: MBG/26SCS bracket is a large amount of by porous structure physical absorption
26SCS bracket displaing amaranth；And with the introducing of surface amino groups, MBG-NH2/ 26SCS bracket shows more strong purplish red
Color colour developing, shows amination modified bracket MBG-NH2Facilitate higher and more stable 26SCS load.
Test example 3
BMP-2 (load capacity are as follows: 1 μ g) and 26SCS (load capacity are as follows: 2 μ g) are born using negative-pressure adsorption-desivac
It carries, MBG-NH2The quality of bracket is 0.075g, and the release of BMP-2 is quantitative determined by enzyme linked immunosorbent assay.Experimental procedure is such as
Under: load is had to the activity glass bracket MBG-NH of BMP-2 and 26SCS2/ BMP-2/26SCS (cylindrical stent size: diameter
6mm, height 2mm) it is put into 2mL PBS buffer solution (pH=7.4), it is cultivated in 37 DEG C of vibration culture ovens；It, will in preset time point
2mL PBS all takes out and changes to fresh PBS buffer solution, using releasing for Human BMP-2 ELISA Kit quantitative determination BMP-2
To one's heart's content condition；It selects to be handled without amination with the MBG/BMP-2 bracket of 26SCS load as a control group in experiment.Due to liver
On element electronegative N- and O- sulfonic acid group can on BMP-2 lysine and arginine residues in conjunction with, and as one
Kind of heparinoid polysaccharide, 26SCS have a structure similar with heparin, and with higher elecrtonegativity N- and O- sulfonic acid group with
The electrostatic interaction of BMP-2 generation three-dimensional effect.Therefore, after BMP-2 is in conjunction with the 26SCS on bracket, BMP-2 can effectively be slowed down
Diffusion.As tested in Fig. 8 as the result is shown: the protein delivery behavior of two kinds of brackets is shown typically: (24 is small for early stage burst release
When it is interior) and the later period sustained release (240 hours).Both comparisons release profiles, MBG-NH2/ BMP-2/26SCS bracket can be effective
Slow down the diffusion of BMP-2, shows gentler release behavior in the entire experiment process.
Test example 4
MBG-NH is measured using CCK-8 method2/ BMP-2/26SCS bracket increases hBMSCs (human marrow mesenchymal stem cell)
The influence grown: by logarithmic growth phase cell dissociation, (2.0 × 10 are counted4Cells/sample (bracket ruler is inoculated on bracket after)
Very little same test example 3).Culture discards original fluid after a certain period of time, and the fresh culture of the solution containing 10%CCK-8 is added in every hole,
With absorbance OD value at enzyme-linked immunosorbent assay instrument detection 450nm wavelength after 37 DEG C of culture 2h.In experiment with MBG/BMP-2 bracket and
Simple MBG bracket is as a control group.As a result as shown in figure 9, cell rack surface have good growth tendency, OD value with
Time and increase；And with the extension of incubation time, the group containing BMP-2 load: MBG/BMP-2 and MBG-NH2/BMP-2/
Cell Proliferation of the 26SCS in 3d and 7d has significant difference compared with MBG group, wherein MBG-NH2/ BMP-2/26SCS bracket
BMP-2 is enabled to continuingly act on BMSCs the sustained release of BMP-2, proliferation is the most significant compared with MBG.
Test example 5
It is living by detection of alkaline phosphatase (ALP, osteoblast mineralization play the functional activity enzyme of main function in the process)
Property investigates the ability of stent-induced cell Osteoblast Differentiation.Specific step is as follows: by cell with 5.0 × 104Cells/well's
Density is inoculated in 48 orifice plates, and and MBG-NH2/ BMP-2/26SCS bracket (stent size is with test example 3) co-cultures；Cultivate 3d
With culture solution is discarded after 7d, by cell under orifice plate with PBS clean three times, be added 200 μ L 1%Triton X-100 lysates, split
Solve cell 2h；It takes 50 μ L supernatants to be added in 96 new orifice plates after cell pyrolysis liquid centrifugation, and 200 μ L is added to every hole
PNPP solution is in 37 DEG C of incubation 2h；After incubation, enzyme-linked immunosorbent assay instrument measures the ALP light absorption value at 405nm；ALP activity
For the ratio of light absorption value at 405nm and corresponding total protein, the content of total protein is by BCA kit measurement in lysate.In experiment
Selection blank orifice plate (Blank well), BMP-2, MBG bracket and MBG/BMP-2 bracket are as a control group.As a result such as Figure 10
Shown, the ALP expression of the addition of no BMP-2, MBG/26SCS bracket is almost the same with blank well board group；And drawing with BMP-2
Enter, ALP expression up-regulation, but free state BMP-2 half-life short, incubation easy in inactivation, ALP expression quantity when cultivating 3d are
ALP expression is significantly reduced to suitable with blank group after 1.88,7d.In comparison, MBG/BMP-2 and MBG-NH are incubated at2/BMP-
Cell ALP activity on 2/26SCS bracket is significant to be increased, and it is free that the expression quantity of each group, which is respectively 4.80 and 5.42, when 3d
2.55 and 2.88 times or more of BMP-2 group；When culture incubation time extends to 7d, MBG-NH2/ BMP-2/26SCS rack surface
The ALP activity of cell is still significantly higher than remaining each group, and 1.65 and 2.8 times of respectively MBG/BMP-2 and free BMP-2 group,
It shows and excellent facilitates bone differentiation capability.Demonstrating 26SCS can stablize and improve BMP-2 vigor, to show more
Osteogenic differentiation activity outstanding.Simultaneously by 5.0 × 104Cells/sample cell inoculation uses after cultivating 3d in bracket
BCIP/NBT alkaline phosphatase colour reagent box is to MBG/BMP-2 and MBG-NH2Cell on/BMP-2/26SCS bracket into
Row staining analysis, cell dyeing result be consistent with ALP active level testing result, it was demonstrated that 26SCS can stablize and improve BMP-2
Vigor, so that activity glass bracket shows higher osteogenic differentiation activity.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of polyose modification MBG bracket, which is characterized in that the polyose modification MBG bracket includes MBG bracket and is supported on
Polysaccharide derivates on MBG bracket.
2. polyose modification MBG bracket according to claim 1, which is characterized in that the MBG bracket is three-dimensional porous connection
Network structure, has 200-500 μm of connection macropore, and 0.5-5 μm of surface micropore and aperture are the order mesoporous of 6-9nm；
Preferably, the skeleton hole wall thickness of the MBG bracket is 40-100 μm.
3. polyose modification MBG bracket according to claim 1, which is characterized in that be modified with amino on the MBG bracket.
4. polyose modification MBG bracket according to claim 1-3, which is characterized in that the polysaccharide derivates packet
Include at least one of sulfated chitosan, hyaluronic acid, chondroitin sulfate, Heparan sulfate or heparin, preferably sulfonation shell
Glycan；
The polysaccharide derivates quality be MBG bracket quality 0.001%-0.3%, preferably 0.003%.
5. the preparation method of any one of the claim 1-4 polyose modification MBG bracket, which is characterized in that by polysaccharide derivates
It is carried on MBG bracket, obtains polyose modification MBG bracket.
6. preparation method according to claim 5, which is characterized in that by polysaccharide derivates negative-pressure adsorption on MBG bracket,
Freeze-drying obtains polyose modification MBG bracket.
7. preparation method according to claim 5 or 6, which is characterized in that further include first repairing MBG bracket progress amination
Decorations processing, the step of then polysaccharide derivates are carried on MBG bracket again, obtain polyose modification MBG bracket；
Preferably, the step of amination moditied processing includes: to mix MBG bracket, low-carbon alcohols and amino silicane coupling agent
24-96h obtains amido modified MBG bracket, wherein MBG bracket quality g, low-carbon alcohols volume ml and amino silicane coupling agent volume
The ratio of ml is 1:150-250:3-8；
Preferably, the amino silicane coupling agent includes gamma-aminopropyl-triethoxy-silane, γ-aminopropyltrimethoxysilane
Or N- β (aminoethyl)-γ-aminopropyltrimethoxysilane, preferably gamma-aminopropyl-triethoxy-silane；
Preferably, the low-carbon alcohols include methanol, ethyl alcohol, propyl alcohol, isopropanol, butanol, isobutanol, preferably ethyl alcohol, further
Preferably dehydrated alcohol；
Preferably, the method also includes washing again after MBG bracket, low-carbon alcohols and amino silicane coupling agent mixing 24-96h
The step of washing；
Preferably, the step of washing includes: independently to be washed 3 times with ethyl alcohol and ultrapure water, obtains amino after dry and repairs
Adorn MBG bracket；
Preferably, by slurry cast to template, removing template is removed after dry, obtains the MBG bracket, wherein the slurry packet
The mixing of MBG microballoon, micropore pore-forming reagent and MBG colloidal sol is included, the mass ratio of MBG microballoon, micropore pore-forming reagent and MBG colloidal sol is
0.5-1:0.25-1:1-7；
Preferably, the stock quality g and template volume cm3Ratio are as follows: 0.3-0.6:1；
Preferably, the micropore pore-forming reagent includes that polyacrylic acid microballoon, polystyrene microsphere, polymethyl methacrylate are micro-
Ball；
Preferably, the partial size of the polyacrylic acid microballoon is 5-30 μm；
Preferably, the viscosity of the MBG colloidal sol is 4-6 × 104mPa·s；
Preferably, the partial size of the MBG microballoon is 8-12 μm；
Preferably, the MBG microballoon is dry by MBG colloidal sol and ball milling sieving is prepared；
Preferably, the template is polyurethane sponge or polyvinylalcohol sponge；
Preferably, the template is by pretreatment, and the pretreatment includes: to clean 1.5-2.5h with NaOH solution, wherein NaOH
The mass fraction of solution is 10-20%；
Preferably, removing template is gone by the method for sintering, the sintering temperature is 550-650 DEG C, sintering time 5-7h.
8. any one of any one of the claim 1-4 polyose modification MBG bracket or claim 5-7 preparation method obtains
Polyose modification MBG bracket in following A)-D) application in any one:
A) growth factor loads；
B) growth factor slow-release；
C) growth factor activity regulates and controls；
D) tissue renovation material and its preparation.
9. a kind of tissue recovery support, which is characterized in that including the described in any item polyose modification MBG brackets of claim 1-4
Or the polyose modification MBG bracket that is prepared of the described in any item preparation methods of claim 5-7 and it is carried on polyose modification MBG
The growth factor of bracket；
Preferably, the growth factor includes at least one of BMP-2, VEGF or FGF, preferably BMP-2；
The growth factor quality be polyose modification MBG bracket quality 0.001%-0.1%, preferably 0.0015%.
10. the preparation method of tissue recovery support described in claim 9, which is characterized in that by life in factor negative-pressure adsorption in more
On sugar-modified MBG bracket, freeze-drying obtains tissue recovery support.
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