CN108926745B - A kind of preparation method and its compound system of nanoporous micro rack - Google Patents
A kind of preparation method and its compound system of nanoporous micro rack Download PDFInfo
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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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- 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
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular 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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
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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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- 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
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
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- A—HUMAN NECESSITIES
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- 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
- 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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- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
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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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
Abstract
The invention discloses the preparation method and its compound system of a kind of nanoporous micro rack, the nanoporous micro rack is the three-dimensional porous micro rack of nano-carrier-sodium alginate.The preparation method comprises the following steps: (1) synthesis of mesoporous silica nano-particle；(2) nano-carrier-sodium alginate three-dimensional porous rack preparation；(3) preparation of the three-dimensional porous micro rack of nano-carrier-sodium alginate.The present invention is by the way that the nano-carrier for being loaded with bioactive substance to be coated in sodium alginate porous support, utilize liquid nitrogen flash freezer crush method, develop a kind of compound micro rack system of nano-carrier-sodium alginate, the system have can efficiently promote expansion of stem cells and directed differentiation, it is easy to operate, the features such as Clinical practicability is strong.
Description
Technical field
The present invention relates to a kind of preparation method of bracket and its compound systems, belong to regenerative medicine field, in particular to one
The preparation method and its compound system of kind nanoporous micro rack.
Background technique
Biological support by simulation human body natural microenvironment physicochemical property, can effectively facilitate stem cell self-renewing,
Proliferation and directed differentiation etc., are widely used in regenerative medicine field in recent years.Generally, the biology based on stem cell
Bracket is broadly divided into porous support and hydrogel scaffold by its state.Wherein, porous support has largely interconnected three-dimensional
Porous structure, compared to hydrogel etc., the more conducively supply of migration and nutriment of the cell in bracket.It is generally believed that
100-300 μm of three-dimensional porous structure compares suitable cell and grows into what is organized, therefore porous support is widely applied and is organizing
Regeneration and immunotherapy of tumors research field.
However, porous support traditional at present has biggish physical size, hinders nutriment and cell enters branch
Inside frame, the survival and amplification of cell on bracket have been seriously affected.
For this problem, there is research team to prepare " micro rack " with smaller physical size both at home and abroad.Due to
The physical size very little (about 800-2,000 μm) of micro rack, nutriment and cell can smoothly enter into internal stent, to promote
It survives, be proliferated and the directed differentiation in later period etc. on bracket into cell.For example, Y.N.Du etc. was once reported, they using gelatin and
Polyethylene glycol prepares micro rack respectively to enhance the survival and amplification of liver cell.
But almost all of conventional porous bracket cannot all be transplanted in vivo in a manner of simple injection at present, still
So need to be implanted by traumatic biggish traditional surgery mode.
Injectable biological support is using simplicity and can effectively be sufficient filling with tissue defect, before having biggish clinical application
Scape becomes the forward position research direction of biomaterial in recent years.Current injectable bracket is mostly gel, this kind of bracket knot
Structure is fine and close, hinders the exchange of nutriment/metabolite, while being also unfavorable for the migration of cell.And common porous support
Due to its design feature, it is difficult to be implanted by way of injection.Therefore, it is dedicated to developing there are many researcher in recent years
The porous support of injectable.D.J.Mooney et al. prepares a kind of complex sodium alginate hydrogel scaffold of injectable, the branch
Porous structure can be formed in situ in frame in vivo, to effectively facilitate the stem cell migration that is coated in gel to defect.
MilicaRadisic et al. is designed by structural unit, prepares blood vessel degradable biological chip (the netted knot of single layer of injectable
Structure).
However, not developing yet at present, to effectively facilitate stem cell survival, amplification, the injectable of directed differentiation three-dimensional porous
Micro rack.
Summary of the invention
For overcome the deficiencies in the prior art, it is an object of the present invention to provide a kind of preparation method of nanoporous micro rack,
This method is easy to operate, and gained nano-carrier-three-dimensional porous micro rack of sodium alginate can promote regeneration, can efficiently promote
Expansion of stem cells and directed differentiation, injectable, Clinical practicability are strong.
It is a further object of the present invention to provide a kind of nanoporous micro rack compound system, which contains nanometer load
The three-dimensional porous micro rack of body-sodium alginate, can be applied to regeneration.
In order to achieve the above object, the invention adopts the following technical scheme:
A kind of preparation method of nanoporous micro rack, the nanoporous micro rack are nano-carrier-sodium alginate three
Tie up porous micro rack；The preparation method comprises the following steps:
(1) synthesis of mesoporous silica nano-particle:
Pluronic P-123 (Sigma) 3~9g, 125~130mL of deionized water and dense HCl are added in round-bottomed flask
18~25mL is vigorously stirred 1.5~4h, sets flask in 50 DEG C of water-baths after mixing well, be added with stirring ethyl orthosilicate 5~
10g stirs 20h.Then 80~85 DEG C are warming up to, 24~30h of constant temperature is stood；It is then centrifuged for (5000~6000G), uses deionization
Water washing precipitating, is calcined in Muffle furnace after dry；
(2) nano-carrier-sodium alginate three-dimensional porous rack preparation:
Modified sodium alginate is dissolved in deionized water, is uniformly mixed with mesoporous silica nano-particle；It will mixing
Liquid is placed in 4 DEG C and is placed on -20 DEG C, freeze-drying；Calcium chloride solution is added, PBS is freeze-dried again after embathing, and hermetically drying is low
Temperature saves；
(3) preparation of the three-dimensional porous micro rack of nano-carrier-sodium alginate:
Nano-carrier-sodium alginate three-dimensional porous rack is placed in centrifuge tube, liquid nitrogen is added, will quickly be propped up with homogeneous instrument
Frame is broken；After liquid nitrogen volatilization completely, calcium chloride solution is added, is filtered after reaction, gained particle is freeze-dried,
Up to the three-dimensional porous micro rack of nano-carrier-sodium alginate.
Further, the three-dimensional porous micro rack of nano-carrier-sodium alginate has three-dimensional porous structure, and bracket is ball
Shape, minimum can be made into the microballoon that diameter is 1mm, and aperture can be adjusted according to specific tissue characteristics demand, and pore diameter range is 50~300
μm。
Further, in step (1), the synthetic method of the mesoporous silica nano-particle further includes embedding biological work
Property substance, includes the following steps:
Mesoporous silica nano-particle is added in bioactive substance solution and is reacted, precipitating is bioactive substance
The mesoporous silica nano-particle of modification, is sealed；
The bioactive substance is albumen, polypeptide or small with the maintenance of promotion cell function, proliferation and directed differentiation
Molecule, including but not limited to bone morphogenetic proteins series, vascular endothelial growth factor, oxygen carrier albumen, aspirin, inhomogeneity
Cytokines promote islet cells growth factor, promote liver cell growth factor and promote the Myocyte growth factor.
Preferably, the polypeptide is to promote stem cell skeletonization to differentiation activity substance.
Preferably, the concrete operations of the mesoporous silica nano-particle embedding bioactive substance are as follows: by mesoporous two
Silica nano particle is added in bioactive substance solution, makes its final concentration of 1000 μ g/mL, reacts 1h, is centrifuged on removing
Clear solution, precipitating are the mesoporous silica nano-particle of bioactive substance modification, are centrifuged, will sink after washing 2 times with PBS
It forms sediment and is freeze-dried, seal 4 DEG C of preservations.
Further, in step (2), the modified sodium alginate is that rgd peptide covalence graft modifies sodium alginate, is changed
Property method includes the following steps:
Sodium alginate soln is added in EDC/NHS, after 4h is stirred at room temperature, is added after rgd peptide stirs evenly in 4 DEG C of static mistakes
Night；Then it is freeze-dried for 24 hours after the solution being transferred to bag filter (3.5kDa) dialysis 3d；Sealing, -20 DEG C of storages.
Further, step (2) concrete operations are as follows: modified sodium alginate is completely dissolved in deionized water, makes its end
Concentration is 1.5% (w/v), is uniformly mixed with the mesoporous silica nano-particle of bioactive substance modification；Mixed liquor is turned
Mold (such as agent plate, glass dish) is moved to, 4 DEG C of 1~4h of placement are placed in, is placed on -20 DEG C of 8~20h of placement, freeze-drying 24
~48h；The calcium chloride solution of final concentration of 2% (w/v) is added, is crosslinked 15min, PBS is freeze-dried 12h after embathing 3 times again,
4 DEG C of hermetically drying preservations.
Further, in step (3), liquid nitrogen additional amount is the 10%~45% of centrifuge tube volume (15mL or 50mL)；Chlorine
Change final concentration of 2% (w/v), 2~15min of cross-linking reaction of calcium solution.
Further, in step (3), use revolving speed used in homogeneous instrument high-speed breakage bracket for 20,000~40,000rpm,
High-speed breakage 30s~2min.
Further, in step (3), the filtering uses the standard screen of 1.2~1.5mm of aperture.
A kind of nanoporous micro rack compound system, the nanoporous being prepared containing preparation method as described above are micro-
Bracket.
Further, be transferred to before syringe further includes that a little low-concentration sodium alginate solution solution is added dropwise in micro rack is abundant
The step of mixing；The preparation method of the compound system is the following steps are included: three-dimensional by the nano-carrier prepared-sodium alginate
Porous micro rack is soaked in PBS, and 4 DEG C, 48h；Then PBS is filtered out, NAC/MS is transferred in 1mL syringe, is infused beyond the Great Wall
Piston is penetrated, is sealed for 4 DEG C after freeze-drying.
Compared with prior art, the invention has the following advantages:
(1) the preparation-obtained three-dimensional porous micro rack of nano-carrier-sodium alginate of the present invention, has high connected ratio
Three-dimensional porous structure has smaller physical size, conducive to the entrance of nutriment, is conducive to growing into for cell and tissue, promotees
Into the adherent of cell, survival and proliferation；
(2) the three-dimensional porous micro rack of the preparation-obtained nano-carrier-sodium alginate of the present invention and its compound system, pass through
The mesoporous silica nano-particle of bioactive substance modification is added, can release bioactive substance in growth phase appropriate
It releases, stimulates tissue differentiation, to promote tissue differentiation and maturation to provide lasting " power ", it is visually referred to as by we
" biological engine "；
(3) the three-dimensional porous micro rack of the preparation-obtained nano-carrier-sodium alginate of the present invention and its compound system, one,
Any histocyte direct injection can not be embedded, after being injected into certain tissue, corresponding histocyte can be grown in micro rack；
Two, the histocyte of a certain tissue is embedded in micro rack, is then injected into corresponding position, and it is raw to accelerate corresponding histocyte
It is long；Three, embedding has medicative albumen, polypeptide or small molecule in micro rack, is injected directly into corresponding tissue, Ke Yihuan
On The Drug Release reduces dosage at sufferer；Four, three of the above can be used in mixed way；
(4) the three-dimensional porous micro rack of the preparation-obtained nano-carrier-sodium alginate of the present invention and its compound system, utilize
Liquid nitrogen flash freezer high-speed breakage method is developed a kind of injectable biological support and compound system, is changed by traumatic biggish
The mode that traditional surgery mode is implanted into can be sufficient filling with using simplicity and effectively tissue defect, have huge face
Bed application prospect.
Detailed description of the invention
Figure 1A is the pictorial diagram of injectable NAC/MS and SEM figure in embodiment 2；
Figure 1B is the physical size of different support in embodiment 2；
Fig. 1 C is the aperture characterization result of different support in embodiment 2；
Fig. 2 is injectable NAC/MS syringeability verification result in fine and close soft tissue in embodiment 2；
Fig. 3 A is the elasticity modulus test result of different support in embodiment 2；
Fig. 3 B is elution profiles of the BFP-1 in different support in embodiment 2；
Fig. 3 C is reservation situation of fluorescent marker (FITC-) BFP-1 in different support in embodiment 2；
Fig. 4 A is hMSCs multiplication rate measurement result (CCK-8) in embodiment 4；
Fig. 4 B is that growth morphology SEM of the hMSCs on each bracket schemes (3 days) in embodiment 4；
Fig. 4 C is the distribution and quantity qualitative determination that hMSCs is grown on each bracket in embodiment 4；
Fig. 4 D is 1 expression of integrin-β (7 days) that hMSCs is grown on each bracket in embodiment 4；
Fig. 4 E is the photograph via bright field (7 days) that hMSCs is grown on each bracket in embodiment 4；
Fig. 5 A is " skeletonization biology engine " schematic diagram in embodiment 4；
Fig. 5 B is external ALP Activity determination result in embodiment 4；
Fig. 5 C is Bone formation-related gene testing result in embodiment 4；
Fig. 5 D is correlative protein expression Westernblot testing result in embodiment 4.
Specific embodiment
Now in conjunction with attached drawing, the invention will be further described with specific embodiment.
The present invention is directed to develop a kind of compound micro rack system of nano-carrier-sodium alginate, efficiently to promote stem cell expansion
Increasing and directed differentiation efficiency, fast implement regeneration and restoration.We are coated on the nano-carrier for being loaded with bioactive substance
In sodium alginate porous support, using liquid nitrogen flash freezer crush method, the three-dimensional porous micro rack body for being loaded with " biological engine " is developed
System.
In order to verify the biology effect of the compound system, we pass through " biological engine " using osteanagenesis reparation as example
Bionical release skeletonization polypeptide BFP-1 promotes hMSCs Osteoblast Differentiation and maturation.
The injectable NAC/MS preparation method that result of study shows that we develop is simple, easy to use, has very big
Value for clinical application, which also has the potentiality applied to hepatic tissue regeneration and the fields such as myocardial repair.
The present invention develops a kind of compound micro rack system of nano-carrier-sodium alginate, which has and can efficiently promote to do
The features such as cell expands and directed differentiation, easy to operate, and Clinical practicability is strong.
Particular content of the present invention is as follows:
One, injectable nano-carrier-three-dimensional porous micro rack of sodium alginate (NAC/MS) preparation and characterization:
Centrifuge tube is added in liquid nitrogen, submerges uncrosslinked nano-carrier-sodium alginate three-dimensional porous rack (NACS), toughness
Biggish bracket will become very brittleness, and homogeneous instrument high speed rotation will generate biggish shearing force, finally will be larger-size
Bracket is broken into very small size of porous particle, these particles are exactly obtained three-dimensional after being crosslinked and be lyophilized using calcium chloride
Porous micro rack (NAC/MS).
Compared to the other modes (for example being sheared with scissors, blade) for preparing the micro rack, liquid nitrogen is instantaneously ultralow in this method
Temperature can quickly fix the porous structure of NACS (or AS), so that will not be destroyed by subsequent shearing force.
In addition, this method operates at low temperature, the activity that biotic factor is coated in bracket will not influence.
In order to make micro rack have syringeability, the micro rack of freeze-drying is immersed in PBS by we, it is therefore an objective to remove micro rack
Extra Ca remained on surface2+, after its abundant water absorption and swelling, a little low concentration alginic acid is added dropwise in micro rack after filtering PBS
Sodium solution mixes well, and transfers to freeze-drying in syringe (1mL specification).When experiment in vivo, cell suspension need to only be added
Mixing well into micro rack can be injected.
The sodium alginate soln of dropwise addition plays certain " lubrication ", and micro rack particle can be made to pass through injection needle.By
It is lower in the concentration of sodium alginate soln, it will be fallen quickly by further dilution after being injected into animal body, will not influence in bracket
The cell activity of inoculation.
The gained three-dimensional porous micro rack of nano-carrier-sodium alginate of the invention has a three-dimensional porous structure, bracket be it is spherical,
Minimum can be made into the microballoon that diameter is 1mm, and aperture can be adjusted according to specific tissue characteristics demand, and specific pore size is according to certain group
Most suitable aperture needed for knitting growth is adjusted, and controllable range is 50~300 μm.
The three-dimensional porous micro rack of the preparation-obtained nano-carrier-sodium alginate of the present invention and its compound system, one, it can be with
Any histocyte direct injection is not embedded, and after being injected into certain tissue, corresponding histocyte can be grown in micro rack；Two,
The histocyte that a certain tissue is embedded in micro rack is then injected into corresponding position, accelerates corresponding tissue cell growth；
Three, embedding has medicative albumen, polypeptide or small molecule in micro rack, is injected directly into corresponding tissue, can be slow
Release reduces dosage at sufferer；Four, three of the above can be used in mixed way.
Nano-carrier is the carrier of a kind of drug or bioactive substance, its effect is further subtracted on bracket basis
Slow drug or bioactive substance sustained release rate.
It is received to show for BFP-1 " loading " to be coated in again in mesoporous silica nano-particle (MSNs) vividerly
In the three-dimensional porous micro rack of meter Zai Ti-sodium alginate (NAC/MS), than BFP-1 is directly coated in sodium alginate bracket (pep
AMS there is the longer sustained release period, we determine the elution profiles and fluorescence reserved graph of BFP-1 in two kinds of systems in).
The experimental results showed that the BFP-1 in NAC/MS shows obvious slower rate of release, by BFP-1 " dress
Carry " the bionical sustained release process will be effectively realized in MSNs.The BFP-1 of " loading " in NAC/MS, than being directly coated with BFP-1
(pep@AMS) has the longer sustained release period in sodium alginate bracket.BFP-1 in pep@AMS has discharged nearly one in 5d
Half, and the BFP-1 in NAC/MS is until the half of 10d ability total volume.The fluorescence reserved graph of fluorescent marker BFP-1 shows,
BFP-1 in NAC/MS shows obvious slower rate of release, supports the measurement result of elution profiles, it was demonstrated that
MSNs has delay, constantly slow releasing function to biotic factor.
Carrier of the mesoporous silica nano-particle as biotic factor in the present invention, can be according to application background need
It wants, the medicative albumen of a variety of tools of delivery, polypeptide or small molecule, as there is the maintenance of promotion cell function, proliferation and determine
To the albumen of differentiation, polypeptide or small molecule isoreactivity substance, active material includes but is not limited to bone morphogenetic proteins series, blood
Endothelial tube growth factor, oxygen carrier albumen, aspirin, different type cell factor, rush islet cells growth factor, rush liver are thin
The intracellular growth factor and the rush Myocyte growth factor, and the mode long-time to continue, stable is sustained.For example, if this is propped up
Frame is used for myocardial infarction treatment, then " loading " can promote stem cell myocardium differentiation and the mature factor in nano particle, we
The nano particle of the load biotic factor is known as " biological engine ".
Two, the injectable nano-carrier-three-dimensional porous micro rack of sodium alginate (NAC/MS) bone tissue engineer application:
As verifying and demonstration, we by NAC/MS system be applied to bone tissue engineer, using BFP-1 as rush stem cell at
The biotic factor of bone differentiation.
Result of study shows that stem cells proliferation and Osteoblast Differentiation can be promoted using NAC/MS as bracket.NAC/MS energy
It is obviously promoted the ALP activity of hMSCs, the BFP-1 being sustained out has played effect.Further, Bone formation-related gene and albumen table
Shown up to test result, NAC/MS can effectively raise each Bone formation-related gene of hMSCs (including Runx2, OCN, Col1a1, OPN and
ALP expression)；The expression of Westernblotting Runx2, OCN, Col1a1 albumen as the result is shown equally supports base
The conclusion of cause and ALP Activity determination.
The present invention demonstrates the influence that NAC/MS is survived to hMSCs and is proliferated.Promoting stem cells hyperplasia is NAC/MS design
One of critical function, we are intended to by preparing micro rack more smaller than traditional common porous stent size, allow nutriment
Internal stent can more fully be entered with cell, and survival and proliferation rate of the stem cell in porous support are promoted with this.
Below by way of specific preferred embodiment combination attached drawing further illustrate the present invention, but the present invention be not limited in it is following
Embodiment.
The preparation of the three-dimensional porous micro rack of 1 nano-carrier of embodiment-sodium alginate
1.1. peptide modified mesoporous silica nano-particle (peptide-laden mesoporous silica
Nanoparitcles, pep@MSNs) synthesis
Mesoporous silica nano-particle (MSNs) derives from the laboratory East China University of Science Wei Jie.Specific preparation method
Are as follows: be added in round-bottomed flask Pluronic P-123 (Sigma) 3~9g, 125~130mL of deionized water and dense HCl 18~
25mL is vigorously stirred 1.5~4h, sets in 50 DEG C of water-baths flask after mixing well, is added with stirring 5~10g of ethyl orthosilicate,
Stir 20h.Then 80~85 DEG C are warming up to, 24~30h of constant temperature is stood；It is then centrifuged for (5000~6000G), is washed with deionized water
Precipitating is washed, is calcined in Muffle furnace after dry.
In this research, the polypeptide that we use is derived from a segment polypeptide of the non-maturation zone of Bone Morphogenetic Protein-7, is named as
BFP-1 (GQGFSYPYKAVFSTQ shines by force purchased from Shanghai).
10mLBFP-1 solution (10 is added in 100mgMSNs powder-4Mol/L 1h is reacted in), is then centrifuged for separating
(2000rpm, 2min), removes supernatant solution.The precipitating is pep@MSNs, is centrifuged after washing 2 times with PBS, by pellet frozen
It is dry, then seal 4 DEG C of preservations.
1.2. nano-carrier-sodium alginate three-dimensional porous rack (nanocarriers-alginate composites
Scaffolds, NACS) preparation
Sodium alginate (alginate) used in this research is rgd peptide (GGGGRGDASSP shines by force purchased from Shanghai).
The method of modifying of RGD-alginate (RA):
Sodium alginate soln is added in EDC/NHS, after 4h is stirred at room temperature, is added after rgd peptide stirs evenly in 4 DEG C of static mistakes
Night；Then the solution is transferred to be freeze-dried after bag filter (3.5kDa) dialysis 3d and obtains RA for 24 hours；Sealing, -20 DEG C of storages.
2.0g RA is dissolved in 100mL deionized water, pep@MSNs powder is added after completely dissolution to it and is sufficiently mixed
Uniformly.Then mixed liquor is transferred in 24 orifice plates, the hole 0.5mL/, orifice plate is successively then placed in 4 DEG C of refrigerator 2h, -20 DEG C
After refrigerator 8h, freeze-drying is for 24 hours.After the completion of freeze-drying, calcium chloride (2%, w/v) solution is added in orifice plate, the hole 1mL/, is handed over
Join 15min.Then it is embathed 3 times with PBS, is freeze-dried 12h again, hermetically drying is placed in 4 DEG C of preservations.
The porous support of the not mixed pure RA preparation of pep@MSNs powder is common sodium alginate porous support (AS), for control
Group.
1.3. the three-dimensional porous micro rack of nano-carrier-sodium alginate (nanocarriers-alginate composites
Micro-scaffolds, NAC/MS) preparation
NAC/MS is prepared using liquid nitrogen flash freezer porous support and with the method for homogeneous instrument high-speed breakage:
1 NACS (or AS) is put in 50mL centrifuge tube, and 10mL liquid nitrogen is then added, is quickly crushed bracket with homogeneous instrument
(35,000rpm, 1min).After liquid nitrogen volatilization completely, 10mL calcium chloride solution (2%, w/v) is added into centrifuge tube, crosslinking
Then 5min is filtered with the standard screen of 1.2~1.5mm of aperture, particle of the retention diameter in 1.0mm or so.Then by institute
After particle is placed in -20 DEG C of 4~8h of freezing, then be freeze-dried 8~for 24 hours, obtain NAC/MS.
The micro rack as made from AS is known as AMS, as another control group.
The characterization of the three-dimensional porous micro rack of 2 nano-carriers of embodiment-sodium alginate
Scanning electron microscope (SEM；S-4800；Hitachi, Japan) for characterizing the microcosmic of AS, AMS and NAC/MS
Structure (aperture, porous form etc.) and micro rack size；Modulus of elasticity in comperssion tester (ElectroForce 3100；
Bose, USA) for measuring the elasticity modulus of each bracket.
The micro rack of freeze-drying is immersed in PBS, after its abundant water absorption and swelling, is added dropwise in micro rack after filtering PBS
A little low-concentration sodium alginate solution solution mixes well, and transfers to freeze-drying in syringe (1mL specification).When experiment in vivo,
Cell suspension need to only be added in micro rack and mix well and can inject.Its syringeability is verified with fine and close tissue.
It is coated on elution profiles characterization of the slow release characteristic of the BFP-1 in NAC/MS by measurement BFP-1 at any time.Simply
Ground is used to prepare BFP-1 used in the NAC/MS of measurement sustained release performance by fluorescent tag label mistake, NAC/MS is immersed in
In PBS (37 DEG C), a certain amount of supernatant solution is acquired at the time point preset, utilizes the absorbance of fluorescence microplate reader measurement solution
Value, then by the way that the relation curve of the BFP-1 mass being sustained out in solution at any time is calculated to get to elution profiles.
Experimental result:
As shown in Figure 1A, SEM as the result is shown NAC/MS have high connected ratio three-dimensional porous structure, pore structure also not by
Damage；As shown in Figure 1B-C, the microballoon that stent size is about 1mm, aperture is about 110 μm, which is conducive to cell and group
That knits grows into.
As shown in Fig. 2, the injectable bracket can be easily injected into fine and close tissue, such as beef.
As shown in Figure 3A, the degradation rate of micro rack is apparently higher than AS after three weeks, Ca2+The reason of being crosslinked scaffold degradation
It is because of Ca2+Caused by being exchanged with the univalent cation in solution.The specific surface area of NAC/MS and AMS is significantly larger than AS, therefore
Ca in NAC/MS and AMS2+Exchange rate will be faster than AS, and this difference is significantly showed after three weeks.NAC/
The degradation rate of BFP-1 sustained release rate and bracket in MS has certain relationship, and the degradation of bracket will accelerate the release of BFP-1
Rate.
As shown in Fig. 3 B-C, the BFP-1 in NAC/MS shows obvious slower rate of release, it was demonstrated that MSNs pairs
Biotic factor has delay, constantly slow releasing function.
A large amount of early-stage study discoveries stimulate its directed differentiation will through bionical mode after the stem cell fast breeding phase
It effectively improves the rate of amplification of cell and promotes the directed differentiation of stem cell.
The experimental results showed that BFP-1 " loading " will effectively be realized into the bionical sustained release process in MSNs, the system
Building will lay the foundation for follow-up study.
3 injectable NAC/MS of embodiment preparation
The NAC/MS prepared is soaked in PBS, 4 DEG C, 48h；Then PBS is filtered out, NAC/MS is transferred to 1ml note
In emitter, injection ram, is sealed for 4 DEG C after freeze-drying beyond the Great Wall.
Proliferation of 4 human marrow mesenchymal stem cell of embodiment (hMSCs) on bracket
4.1. cell culture and inoculation
Human marrow mesenchymal stem cell (hMSCs；ScienCell, CA, USA) it is cultivated at DMEM in high glucose (Hyclone, USA)
Amplification in base (10% volume fetal calf serum of addition, 1% volume are dual anti-), and the culture medium is known as proliferated culture medium.
After hMSCs is passaged to the 4th generation (P4), with pancreatin (0.05%trypsin/EDTA, Gibco) digestion, it is centrifuged, again
Each bracket is seeded to after outstanding, inoculation quantity is 1x105The hole cells/.
After being incubated for 2h in incubator, proliferated culture medium is added to every hole 1.5mL.
Second day after inoculation, changes and add Osteogenic Induction Medium, consisting of low sugar DMEM, and add fetal calf serum (10%
Volume), dual anti-(1% volume), β-phosphoglycerol (10mM), ascorbic acid (50ug/mL), dexamethasone (0.1uM).It is all thin
Change a not good liquor within born of the same parents every two days.
4.2.hMSCs proliferation
Use Cell counting Kit (CCK-8；Dojindo, Japan) the 1st, 3,7 and 14 after cell inoculation
It measures the multiplication rate of cell on each bracket.
4.3.hMSCs morphology analysis
The pattern of cell on each bracket is observed using SEM；It is swept with phalloidine marking cytoskeleton and with confocal laser
Retouch microscope (CLSM) observation.
4.4. external skeletonization correlating markings analyte detection
Use the ALP activity in alkaline phosphatase (ALP) activity detection kit (Nanjing is built up) measurement group of cells；It is logical
Cross the Bone formation-related gene expression in real-time PCR instrument measurement group of cells, including a Collagen Type VI (Col1a1), bone
Calcium element (OCN), bone bridge element (OPN), ALP, Runx2, internal reference are β-actin；It is analyzed further combined with Western Blotting
Method detects skeletonization correlative protein expression level, including Col1a1, OCN and Runx2.
Experimental result:
As shown in Figure 4 A, CCK-8 test result shows the multiplication rate with higher of the hMSCs in AMS and NAC/MS,
And after culture 3 days, multiplication rate will be much higher than the hMSCs in AS bracket.The reason is that AMS and NAC/MS are compared to AS
With smaller physical size, as previously described, the porous support of small physical size is more conducive to the entrance of nutriment, promotes
The fast breeding of hMSCs；And the cell in AS, due to cannot get enough nutrition supplies, the third day after cell inoculation is
Show lower multiplication rate.
In addition, we also analyze the adherent form of cell, distribution and quantity etc..As shown in Figure 4 B, AMS and NAC/
HMSCs in MS show it is wider it is flat sprawl form, and the hMSCs in AS then seems more modest.And such result this master
If being determined by the height of cell adherence quality, adherent effect is better, and cell will be sprawled wider flat.
In order to further verify the conclusion, we in each bracket with the adherent performance-relevant integrin β_1 of hMSCs
(integrin β 1) expression is analyzed.
Fig. 4 C is the immunofluorescence analysis of integrin β_1 expression quantity as a result, being seeded in AMS and NAC/ as we can see from the figure
HMSCs ratio in MS has more integrin β_1s to express in AS.It can be with from cell distribution and quantitative analysis result (Fig. 4 C-E)
Find out, the hMSCs in AMS and NAC/MS is evenly distributed, however the hMSCs in AS is only more in edge distribution, and centre is several
Cell-free survival (we term it " edge effects ").
Result above confirms that the micro rack that we prepare can effectively facilitate the adherent of hMSCs, survival and proliferation, and
The introducing of pep@MSNs will not influence hMSCs or more property.
Most crucial part is " skeletonization biology engine " system in NAC/MS, and the working principle diagram of the system is as shown in figure 5.
The pep@MSNs for being loaded with BFP-1 will be released stimulation hMSCs Osteoblast Differentiation in growth phase appropriate, to promote
HMSCs Osteoblast Differentiation and maturation provide lasting " power ", therefore it is visually referred to as " skeletonization biology engine " by we.
ALP is stem cell Osteoblast Differentiation very important marker early period, and activity is higher to show Osteoblast Differentiation degree more
It is high.When Fig. 5 B is shown in the 14th day, NAC/MS can be obviously promoted the ALP activity of hMSCs, and the BFP-1 being sustained out has played effect.
In conjunction with front BFP-1 elution profiles it is found that BFP-1 just plays a role on day 4, therefore NAC/MS and AMS was in the 7th day ALP
Active no difference of science of statistics.
In order to further probe into and verify hMSCs Osteoblast Differentiation degree in each group, we are further to Bone formation-related gene
Detected (Fig. 5 C-D) with albumen, as the result is shown NAC/MS can effectively raise each Bone formation-related gene of hMSCs (including
Runx2, OCN, Col1a1, OPN and ALP) expression；Westernblotting Runx2, OCN, Col1a1 albumen as the result is shown
Expression equally support the conclusion of gene and ALP Activity determination.
The invention is not limited to above embodiment, if not departing from the present invention to various changes or modifications of the invention
Spirit and scope, if these modification and variations belong within the scope of claim and equivalent technologies of the invention, then this hair
It is bright to be also intended to encompass these changes and change.
Claims (9)
1. a kind of preparation method of nanoporous micro rack, it is characterised in that:
The nanoporous micro rack is the three-dimensional porous micro rack of nano-carrier-sodium alginate；
The preparation method comprises the following steps:
(1) synthesis of mesoporous silica nano-particle:
Pluronic P-1233~9g, 125~130mL of deionized water and dense HCl18~25mL are added in round-bottomed flask, it is acute
1.5~4h of strong stirring, sets flask in 50 DEG C of water-baths after mixing well, and is added with stirring 5~10g of ethyl orthosilicate, stirring
20h is then warming up to 80~85 DEG C, stands 24~30h of constant temperature；It is then centrifuged for, precipitating is washed with deionized, in horse after drying
Not calcined in furnace；
(2) nano-carrier-sodium alginate three-dimensional porous rack preparation:
Modified sodium alginate is dissolved in deionized water, is uniformly mixed with mesoporous silica nano-particle；Mixed liquor is set
- 20 DEG C are placed in 4 DEG C, freeze-drying；Calcium chloride solution is added, PBS is freeze-dried again after embathing, and hermetically drying saves；
(3) preparation of the three-dimensional porous micro rack of nano-carrier-sodium alginate:
Nano-carrier-sodium alginate three-dimensional porous rack is placed in centrifuge tube, liquid nitrogen is added, is quickly broken bracket with homogeneous instrument
It is broken；After liquid nitrogen volatilization completely after, be added calcium chloride solution, be filtered after reaction, by gained particle be freeze-dried to get
The three-dimensional porous micro rack of nano-carrier-sodium alginate.
2. preparation method according to claim 1, it is characterised in that: the nano-carrier-sodium alginate is three-dimensional porous micro-
Bracket has three-dimensional porous structure, and bracket is microballoon, and minimum can be made into the microballoon that diameter is 1mm, and aperture is special according to specific tissue
Property demand adjustment, pore diameter range be 50~300 μm.
3. preparation method according to claim 1, which is characterized in that in step (2), the modified sodium alginate is RGD
Polypeptid covalence grafting and modifying sodium alginate, method of modifying include the following steps:
Sodium alginate soln is added in EDC/NHS, after 4h is stirred at room temperature, is added after rgd peptide stirs evenly in 4 DEG C of left undisturbed overnights；
Then it is freeze-dried for 24 hours after the solution being transferred to bag filter dialysis 3d；Sealing, -20 DEG C of storages.
4. preparation method according to claim 1, it is characterised in that: the concrete operations of step (2) are as follows: by modified alginic acid
Sodium is completely dissolved in deionized water, makes its final concentration of 1.5%w/v, the mesoporous silicon oxide with bioactive substance modification
Nano particle is uniformly mixed；Mixed liquor is placed in 4 DEG C of 1~4h of placement, is placed on -20 DEG C of 8~20h of placement, freeze-drying 24~
48h；The calcium chloride solution of final concentration of 2%w/v is added, is crosslinked 15min, PBS is freeze-dried 12h again after embathing 3 times, seal
Dry 4 DEG C of preservations.
5. preparation method according to claim 1, it is characterised in that: in step (3), liquid nitrogen additional amount is centrifuge tube volume
10%~45%；The final concentration of 2%w/v of calcium chloride solution, 2~15min of cross-linking reaction；Using homogeneous instrument high-speed breakage branch
Revolving speed used in frame is 20,000~40,000rpm, high-speed breakage 30s~2min.
6. preparation method according to claim 1, it is characterised in that: in step (3), it is described filtering using aperture 1.2~
The standard screen of 1.5mm.
7. a kind of nanoporous micro rack compound system, it is characterised in that: the compound system contains any one of claim 1-6
The nanoporous micro rack that the preparation method is prepared.
8. nanoporous micro rack compound system according to claim 7, which is characterized in that the preparation of the compound system
Method the following steps are included:
The three-dimensional porous micro rack of nano-carrier-sodium alginate prepared is soaked in PBS；PBS is filtered out, nanometer is carried
The three-dimensional porous micro rack compound system of body-sodium alginate is transferred in syringe, beyond the Great Wall injection ram, is sealed and is protected after freeze-drying
It deposits.
9. nanoporous micro rack compound system according to claim 8, it is characterised in that:
Further include the steps that a little low-concentration sodium alginate solution solution is added dropwise in micro rack to be mixed well before being transferred to syringe；
The temperature being soaked in PBS is 4 DEG C, time 48h；Then PBS is filtered out, by nano-carrier-sodium alginate three
It ties up porous micro rack compound system to be transferred in 1mL syringe, beyond the Great Wall injection ram, be sealed for 4 DEG C after freeze-drying.
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