CN110507861A - It is a kind of can be by nano-particle modified more micropore cytoskeletons and preparation method thereof - Google Patents

It is a kind of can be by nano-particle modified more micropore cytoskeletons and preparation method thereof Download PDF

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CN110507861A
CN110507861A CN201910800722.8A CN201910800722A CN110507861A CN 110507861 A CN110507861 A CN 110507861A CN 201910800722 A CN201910800722 A CN 201910800722A CN 110507861 A CN110507861 A CN 110507861A
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micropore
cytoskeletons
nano
chitosan
agarose
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王彬
喻风雷
陈晓凤
唐敬群
刘文亮
陈名久
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Second Xiangya Hospital of Central South University
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Second Xiangya Hospital of Central South University
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • 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
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    • 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
    • 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
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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
    • A61L2300/20—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
    • 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
    • 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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Abstract

The invention belongs to organizational project Regeneration and Repair field, open one kind can be by nano-particle modified more micropore cytoskeletons and preparation method thereof and nano-particle modified more micropore cytoskeletons.More micropore cell scaffold materials of the present invention are made of chitosan, gelatin and agarose.Chitosan, agarose and gelatin three prepare more micropore cytoskeletons of the interconnection microvia that section is in 100 μm of sizes and the irregular honeycomb appearance structure that thin-walled is constituted by being chemically crosslinked freezing gel method.More micropore cytoskeletons of the present invention can be by nano particle chemical modification.Nano-particle modified more micropore cytoskeletons of the present invention have excellent cell factor load capacity, cytokine profiles can efficiently be loaded, and sustained release cell factor 15 days or more after cell factor load, it is expected to the performance biological effect in tissue local continuous and effective.

Description

It is a kind of can be by nano-particle modified more micropore cytoskeletons and preparation method thereof
Technical field
The invention belongs to organizational project Regeneration and Repair fields, and in particular to one kind can be thin by nano-particle modified more micropores Born of the same parents' bracket and preparation method thereof and nano-particle modified more micropore cytoskeletons, especially chitosan oligosaccharide/heparin nano particle The more micropore cytoskeletons of gelatine-chitosan-agarose of modification.
Background technique
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is that most important kind of bone tissue engineer is careful Born of the same parents.Compared with traditional allogeneic MSC transplantation strategies, the mobilization of self MSCs can transplant to avoid Allogeneic stem cell draws The risk of the immunological rejection and transmission of infection that rise.Going back to the nest for MSCs is stimulated by cytokine profiles, proliferation and differentiation are bone groups Knit the only way which must be passed of reparation and regenerative process.Stromal cell derived factor-1 (Stromal cell-derived factor-1, SDF-1 it is) most important chemotactic factor (CF) in stem cell mobilization, plays a crucial role in the mobilization of MSCs.However, bone tissue is again It is raw to repair in addition to MSCs is mobilized, further relate to a series of bioprocess, including MSCs Osteoblast Differentiation and extracellular matrix The synthesis of (extracellular matrix, ECM).Therefore other than chemotactic mobilizes the SDF-1 of MSCs, it is also necessary to another thin The Osteoblast Differentiation of intracellular cytokine promotion MSCs.Bone Morphogenetic Protein-2 (Bone morphogenic protein 2, BMP-2) has promotion Ability of the mescenchymal stem cell (MSCs) to skeletonization lineage.SDF-1 can be raised intracorporal by efficient chemotaxis Mescenchymal stem cell (MSCs) makes target site have the seed cell of enough numbers with for Regeneration and Repair, can be with BMP-2 Synergistic effect is played, osteanagenesis reparation is more effectively promoted.However, these albumen are difficult to repair in bone defect due to half-life short Its bioactivity of the performance of continuous and effective in multiple.For the therapeutic effect of regeneration, pursues and use continual and steady agent Amount delivering is most directly effective strategy.Therefore, a set of load medicine system that can constantly discharge these bioactive molecules is designed System makes cell factor play physiological action in specific organization for a long time and breaks up and increase to promote MSCS going back to the nest in target site It grows.
In recent years, domestic and foreign scholars' research of especially cell factor drug-loading system in terms of drug-loaded biological material achieves Many achievements.There are mainly two types of directions at present, and one is microballoon or nano particle drug-loading systems；One is bracket or gel-likes Drug-loading system.The load capacity of two kinds of drug-loading systems is all very high, but also has the shortcomings that respective.Simple microballoon or nano drug-carrying Although system has large specific surface area, the strong advantage of load capacity, but due to nanoparticle relative distribution and be not fixed, in administration It is easy afterwards with the dynamic diffusion of body fluid flow, it is difficult to guarantee playing a role in fixed target site for continuous and effective.Bracket or gel-like Although drug-loading system overcomes the shortcomings that nano particle is easy diffusion, but also have clearly disadvantageous.Firstly, this kind of drug-loading system is negative Carry cell factor be must bracket or gel preparation when drug is added.And the certain high temperature of preparation process or chemical reaction process It is likely to result in cell factor inactivation, and is difficult to verify its activity after the completion of preparing.Secondly, the drug of this kind of drug-loading system is released Put the degradation speed of the material that places one's entire reliance upon itself, it tends to be difficult to meet actual demand.Both how to overcome the shortcomings of, make cell The factor can be the key scientific problems for needing to solve in the performance bioactivity of the stable continuous and effective of target position.
Summary of the invention
In view of this, can be by nano particle it is an object of the invention to provide one kind aiming at the problems existing in the prior art More micropore cytoskeletons of modification and preparation method thereof and nano-particle modified more micropore cytoskeletons, especially shell are few The more micropore cytoskeletons of the nano-particle modified gelatine-chitosan-agarose of sugar/heparin.
To achieve the purpose of the present invention, the present invention adopts the following technical scheme:
A kind of more micropore cell scaffold materials, are made of chitosan, gelatin and agarose, the gelatine-chitosan-agar Sugared content is 4~5wt%, and the mass ratio of the chitosan, gelatin and agarose is 1:1:(2~3).
Preferably, the porosity of more micropore cell scaffold materials be 79~82%, swelling ratio be 10.36~ 15.03, liquid flow rate > 10mlmin-1。
The present invention also provides the preparation method of more micropore cell scaffold materials, more micropore cell scaffold materials It is prepared by chitosan, gelatin and agarose using chemical crosslinking freezing gel method.Chitosan, agarose and gelatin three are logical It crosses chemical crosslinking freezing gel method and prepares the interconnection microvia that section is in 100 μm of sizes and the irregular honeycomb shape that thin-walled is constituted More micropore cytoskeletons of looks structure.
In some embodiments, the preparation method is specially that gelatin is added after chitosan is dissolved completely in acetum Mix well dissolution, agarose solution be added and mixes, and be added dropwise in blending process 5% (v/v) glutaraldehyde solution into Row crosslinking, is subsequently placed in be incubated at -14 DEG C and prepares within 16 hours freezing gel, thaw at RT prepares more micropore cytoskeletons.
In some embodiments, based on mg/ml, the mass volume ratio of the gelatin and described 5% (v/v) glutaraldehyde solution For 150:1 to 250:1.
The present invention also provides a kind of nano-particle modified more micropore cytoskeletons, by positively charged and can be chemical The nano particle of crosslinking is combined into the more micropore cell scaffold materials of gelatine-chitosan-agarose.
Wherein, the nano particle is positively charged and can be by the nano particle of glutaraldehyde cross-linking.
In some embodiments, the nano particle of the more micropore cytoskeletons of modification is chitosan oligosaccharide/heparin nanometer Grain.
Preferably, the chitosan oligosaccharide/heparin nano particle average particle size range is 100.7~168.2nm, it is average PDI is 0.116~0.122, and average zeta current potential is 24.2mV~33.4mV.
Preferably, it is that chitosan oligosaccharide and heparin pass through that the chitosan oligosaccharide/heparin nano particle preparation method, which is molecular weight, Self-assembly method is made and can be stable at chitosan oligosaccharide/heparin nano particle of physiological pH solution.
Preferably, the molecular weight of the chitosan oligosaccharide is 5000Da；The molecular weight of the heparin is 8000Da；The shell is few The content ratio of sugar and heparin is 16:1.
The present invention also provides the preparation methods of nano-particle modified more micropore cytoskeletons, by the nanometer Grain obtains nano granule suspension after being resuspended in water, and nanometer is added in the more micropore cell scaffold materials of gelatine-chitosan-agarose It is shaken 6 hours for 37 DEG C in grain suspension, 37 DEG C of reaction 20min of glutaraldehyde solution of 5% (v/v) is added, freezed after cleaning dry It is dry.
Preferably, the oscillation rate is 60rpm.
The present invention also provides more micropore cytoskeletons nano-particle modified made from above-mentioned preparation method.
As shown from the above technical solution, the present invention provides it is a kind of can by nano-particle modified more micropore cytoskeletons and Preparation method and nano-particle modified more micropore cytoskeletons.More micropore cell scaffold materials of the present invention are gathered by shell Sugar, gelatin and agarose are made.Chitosan, agarose and gelatin three prepare section by chemical crosslinking freezing gel method The more micropore cytoskeletons for the irregular honeycomb appearance structure that the interconnection microvia and thin-walled of 100 μm of sizes are constituted.Institute of the present invention Stating more micropore cytoskeletons can be by nano particle chemical modification.Nano-particle modified more micropore cytoskeletons of the present invention With excellent cell factor load capacity, cytokine profiles can be efficiently loaded, and are continued after cell factor load Release cell factor 15 days or more, it is expected to the performance biological effect in tissue local continuous and effective.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described.
Fig. 1 show more micropore cytoskeletons macroscopic appearance (A) and SEM image (B, 400 ×, bar=200 μm)；Scanning electricity More micropore cytoskeletons section shows the irregular class cellular structures of thin-walled under mirror；
Fig. 2 shows more micropore cytoskeleton degradation curves, the more micropore cytoskeletons of A group and the more micropore cytoskeleton difference of B group It is incubated for 8 weeks at 37 DEG C of sterile PBS；A group degradation rate is slightly above B group but final palliating degradation degree without marked difference；
Fig. 3 shows by the SEM image of CSO/H nano-particle modified more micropore cytoskeletons；(A, B) is repaired with nano particle The A pack support of decorations；(C, D) is with nano-particle modified B pack support；Micropore cytoskeleton image more than two groups is identical in amplification After multiple (A, C:10000 ×, bar=5 μm；B, D:25000 ×, bar=3 μm), it can be seen that the more micropore cytoskeletons of A group The nanoparticle density of surface modification is apparently higher than B group；Two groups of nano particle is in microspheroidal, and it is thin to be uniformly distributed in more micropores Born of the same parents' rack surface；
Fig. 4, which shows, to be detected using enzyme-linked immunosorbent assay (ELISA) from nano-particle modified more micropore cytoskeletons SDF-1 and BMP-2 release profiles, SDF-1 early stage is in discharge and gradually tend to be steady after outburst release the 4th day, BMP-2 release Trend is slow compared to more flattening out for SDF-1, but involves burst size more than SDF-1 (n=3) in the 10th day beginning BMP-2.
Specific embodiment
The invention discloses a kind of nano-particle modified more micropore cytoskeletons.Those skilled in the art can use for reference this Literary content, is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to art technology It is it will be apparent that they are considered as being included in the present invention for personnel.Method and product of the invention has passed through preferably Embodiment is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to side as described herein Method is modified or appropriate changes and combinations, carrys out implementation and application the technology of the present invention.
For a further understanding of the present invention, below in conjunction with the embodiment of the present invention, to the technical side in the embodiment of the present invention Case is clearly and completely described, it is clear that and described embodiments are only a part of the embodiments of the present invention, rather than all Embodiment.Based on the embodiments of the present invention, those of ordinary skill in the art institute without making creative work The every other embodiment obtained, shall fall within the protection scope of the present invention.
Unless otherwise specified, reagent involved in the embodiment of the present invention is commercial product, can pass through business canal Road purchase obtains.Water used is deionized water.The chitosan oligosaccharide/heparin nano particle use application No. is 201510235782.1 Chinese patent disclosed in method preparation.Using 6 (GraphPad of GraphPad Prism version Software, La Jolla, CA, USA) statistics software carries out statistical analysis, and all results think the difference of value < 0.05 P With statistical significance.The result of all measurement datas all uses mean ± standard deviation to indicate.Two groups and the use of multiple groups measurement data Student ' s-test and One-way ANOVA analysis.
The preparation and characterization of embodiment 1, more micropore cytoskeletons
1, the more micropore cell scaffold materials of gelatine-chitosan-agarose are prepared using chemical crosslinking freezing gel method.
Use gelatin as substrate, is crosslinked using 5% (v/v) glutaraldehyde as chemical cross-linking agent and chitosan, and agar Sugar has the property from gel at low temperature, and triplicity prepares more micropore cytoskeletons.
(1) acetum of 5ml 1% (v/v) is added in 50ml centrifuge tube, electronic balance weighing 100mg chitosan will It is added in above-mentioned acetum, fullys shake, makes it completely dissolved.
(2) it after chitosan is dissolved completely in acetum, weighs 100mg gelatin powder and is added thereto, protected in adition process Hold centrifuge tube shake.40 DEG C of freeze-day with constant temperature of oven are placed in after shaking up to sterilize oven 30 minutes.Making chitosan and gelatin, it is sufficiently mixed It is even and dissolve.
(3) it weighs quantitative agarose powder (A group 200mg, B group 300mg) and is placed in 5ml distilled water (ddH2O), micro-wave oven It is heated to it and sufficiently dissolves (solution becomes clear and shows sufficiently to dissolve).
(4) it after agarose solution being cooled to 50-60 DEG C at room temperature, is added in chitosan-gelatin solution and fullys shake It mixes, (v/v) glutaraldehyde solution of 0.5ml 5% is added dropwise in blending process for being chemically crosslinked.After mixing immediately by solution It pours into 5ml plastic injector.
(5) it is placed in be incubated for 16 hours under -14 DEG C of temperature (cryostat) and prepares freezing gel.Later, by freezing gel It thaws in the deionized water of room temperature, and is taken out from plastic injector mold and prepare more micropore cytoskeletons.To be mostly micro- Hole cytoskeleton is impregnated and is equipped in the beaker of 150ml deionized water, and low speed is washed overnight on the oscillator to rinse crosslinking The unreacted aldehyde radical of agent.More micropore cytoskeletons are dried at room temperature for study its physics and chemical behavior and bio-compatible The inspection of property.
(6) scanning electron microscope characterizes more micropore cytoskeletons, SEM the result is shown in Figure 1.
Macro geometry (Figure 1A) visible its of more micropore cytoskeletons is semi-transparent, flaxen partially brittle solid material Material does not have significant change through 60 DEG C of basic configurations after sterile oven dried 2 hours and mechanical performance, and soaks through PBS buffer solution Brittleness reduces after profit, and elasticity increases.It is in micropore shape that the section naked eyes of more micropore cell scaffold materials are slightly visible.It is micro- under SEM It sees structure to show (Figure 1B), the interconnection microvia that the microcosmic section of more micropore cytoskeletons is about 100 μm by mean size under Electronic Speculum The irregular honeycomb appearance structure (Figure 1B) constituted with thin-walled.From microscopic appearance, more micropore cytoskeletons show cell The desired characteristics of bracket, three-dimensional, microporous interconnection structure.
2, the characterization of more micropore cytoskeletons
To more micropore cytoskeleton porositys, swelling ratio, liquid flow rate and external degradation rate are determined.
(1) more micropore cytoskeleton porosity measurements:
The dehydrated alcohol for measuring certain volume is added in graduated cylinder, and the volume recorded at this time is V1.By more micropores of well cutting Cytoskeleton sample is completely immersed in 30min in dehydrated alcohol, fills ethyl alcohol completely in more micropore cytoskeletons until in liquid Bubble-free evolution, the ethyl alcohol volume recorded at this time is V2.More micropore cytoskeletons are taken out, recording remaining ethyl alcohol volume is V3.Hole The calculation formula of gap rate (Porosity) is as follows:
Porosity (%)=(V1–V3)/(V2-V3) × 100%
Every group takes three groups of sample measurements respectively, and final result takes its average value.
(2) measurement of more micropore cytoskeleton swelling ratios:
The regular more micropore cell scaffold materials of volume are cut, assay balance weighing is recorded as Wd, it is being totally submerged in 37 DEG C PBS in 30min, keep it completely full by PBS.Timbering material is taken out, is weighed after sucking excess surface liquid with filter paper, is remembered Record is Ws.The calculation formula of swelling ratio (Swelling ratio, %) is as follows:
Swelling ratio (%)=(Ws–Wd)/Wd× 100%
Every group takes three groups of sample measurements respectively, and final result takes its average value.
(3) measurement of more micropore cytoskeleton liquid flow rates
More micropore cytoskeletons that length is 1cm are placed in 5ml syringe bottom, after injection top seals with rubber stopper It is fixed on bracket vertically.37 DEG C of PBS100ml is taken to inject in infusion bag, infusion bag connects the syringe needle of 18G.It will infusion Device be hung on 5ml syringe at level height 1m be allowed to keep constant it is hydraulic.Infusion niidl is penetrated to the rubber stopper of syringe, And infusion apparatus valve is opened wide completely, make to be formed in syringe constant hydraulic.
In the apparatus, PBS will be flowed out via after permeating in more micropore cell scaffold materials from syringe outlet.Collect stream PBS out, takes the amount of liquid of wherein intermediate duration 1min, and measures its volume (ml) at room temperature.Pass through more micropores per minute The liquid volume of cytoskeleton is more micropore cytoskeleton liquid flow rates.Every group takes three groups of sample measurements, final result respectively Take its average value.
(4) degradation in vitro of more micropore cytoskeletons
1. assay balance weighs and records the initial dry weight of more micropore cell scaffold materials.
2. and the ethyl alcohol (20-100%) by being incremented by with concentration gradient is handled to sterilize, and processing is used in each step 10-15 minutes.Finally, material is incubated for 20 minutes in 100% fresh ethanol.
3. more micropore cytoskeleton samples of ethyl alcohol sterilizing are carried out freeze-drying process.
4. then the sample being completely dried and the sterile PBS of 30ml are incubated in centrifuge tube.To have more micropore cytoskeletons It is incubated for eight weeks at 37 DEG C of centrifuge tube of material sample.One group of sample is taken out weekly and is washed with deionized to remove the poly- of explanation Close object.And it weighs after being freeze-dried.Three parts of sample, the average dry weight of two samples is taken to calculate palliating degradation degree (Degreeofdegradation, D.D) (%).
D.D (%)=(WI–WF)/WI× 100%
Wherein D.D is degradation journey rate, WIIt is the average dry weight (initial weight) of sample before being incubated for, WFIt is sample after being incubated for Average dry weight (final weight).
3, the parameter characterization result of more micropore cytoskeletons
In multiple combinations of different polymer concentrations, when in polymer solution mixture use 1% chitosan, 1% When gelatin and 2-3% agarose three are chemically crosslinked, it was found that it has preferable ginseng with good mechanical stability Number performance (table 1).
The actual parameter of the micropore cytoskeleton more than two groups of (1) two kind of different formulations preparation is as shown in table 1: micropore more than two groups Cytoskeleton all has high porosity, swelling ratio and liquid flow rate, is suitable for cell adherence, infiltration and proliferation.And gelatin is opposite The more micropore cytoskeletons of the higher A group of content are all apparently higher than B group (p < 0.05) in porosity and swelling ratio.A group degradation rate Slightly above B group (Fig. 2), both more than 10ml/min in terms of the liquid flow rate for representing Permeability Parameters.
Table parameter characterization of micropore cytoskeleton more than 1 two groups and nano particle combination content
* the mass percent of addition solution during gelatine-chitosan-agar sugared content assignment is set in table；# two group pair Than p < 0.05
The combination of embodiment 2, chitosan oligosaccharide/heparin nano particle and more micropore cell scaffold materials
(1) 5mg chitosan oligosaccharide/heparin nano particle is accurately weighed into centrifuge tube, and distilled water dissolution, acetic acid titrates nanometer Grain suspension.
(2) from original more micropore cytoskeleton samples cut into fixed size it is cylindric (diameter: 13 mm, thickness: 2mm), and by assay balance it is weighed and is recorded.It ultrasonic rinsing 5 minutes, is added in nano granule suspension centrifuge tube.
(3) centrifuge tube is placed in 37 DEG C of thermostatic water bath vibrators (60rpm), physics self assembly make nano particle with it is mostly micro- Gelatin mutually adsorbs on the cytoskeleton of hole, is further processed after 6 hours.
(4) 5% glutaraldehyde is added, is crosslinked nano particle further with more micropore cytoskeletons, 37 DEG C of reactions 20min.PBS buffer solution is cleaned 5 minutes × 3 times.
(5) it after the more micropore cell scaffold materials for combining nano particle being carried out freeze-drying process, is received to combining The material of rice grain is weighed.More micropore cytoskeletons is of poor quality to calculate nanometer before and after measuring in conjunction with nano particle The binding capacity of grain.
(6) the combination situation of scanning electron microscopic observation more micropore cytoskeletons and nano particle, scanning electron microscope (Hitachi S-3400N, Japan) obtains image.
The results show that in terms of with chitosan oligosaccharide/heparin nano particle (CSO/H NP) combination, as shown in Fig. 3 and table 1, two The more micropore cytoskeletons of group can be nano-particle modified by CSO/H.In micropore cytoskeleton energy in conjunction with nano particle more than two groups In terms of the comparison of power: the ability of the more micropore cytoskeleton combination nano particles of the more micropore cytoskeleton ratio B groups of A group is stronger.
Since the nano particle binding ability of A pack support has more advantage, select the more micropore cytoskeletons of A group to carry out Subsequent cell factor load efficiency evaluation.
Embodiment 3, cell factor are in conjunction with more micropore cytoskeleton nano surface particles and release in vitro
(1) BMP-2 or SDF-1 are diluted and is configured to various concentration cell factor solution.It will be nano-modified mostly micro- Hole cytoskeleton is totally submerged in cell factor suspension, and 37 DEG C of thermostatic water bath vibrator (60rpm) 6h load it sufficiently. After removing nano-modified more micropore cytoskeletons.By cell factor ELISA kit measure in solution remaining cell because Sub- content, and computational load efficiency.Formula is as follows:
Encapsulation rate=(A-B)/A × 100%
Carrying drug ratio=(A-B)/C × 100%
In formula, A is cell factor gross mass；B is residual cell after the completion of load because of protonatomic mass；C is nano particle matter Amount.
(2) the external release test of cell factor
1. each group is loaded the nano-modified more micropore cytoskeletons of cell factor to be placed in 10mlPBS buffer, it is placed in 37 DEG C, in 60rpm constant water bath box, concussion incubation is carried out in gnotobasis；
2. after being incubated for the scheduled time, nano-modified more micropore cytoskeletons being removed from centrifuge tube and are placed in newly In 10mlPBS buffer.PBS sample originally respectively samples -20 DEG C of freezen protectives after 1ml, temporally with grouping to sample into Line flag；
3. being hereafter sampled according to the time point of setting and updating incubation liquid.It is sequentially placed into the sample cell of number, Freezen protective；
4. the sampling period is 15 days.All samples take out from -20 DEG C of refrigerators, carry out by ELISA detection kit specification Cell factor quantitative analysis calculates the Cumulative release amount of cell factor.
(3) cell factor ELISA test experience
Rewarming is carried out at room temperature using preceding be placed in all reagents and sample.Washing buffer need to be complete to crystalline lens in bottle It is used after the dilution proportion with deionized water by 1:25 (vol:vol) again after dissolution.
1. illustrating to prepare all reagents, working stamndard and sample to be tested according to previous section.It is extra to remove from sheet frame Microwell plate, they are put back in the aluminium foil bag equipped with desiccant pack, is then resealed.
2. 100 μ LAssayDiluent RD1-55 are added into each hole.
3. 100 μ L standard items, reference substance or sample to be tested is added in every hole, sealing is covered with the adhesive tape in kit.Room temperature Under be incubated for 2 hours in the horizontal rail microplate oscillator that revolving speed is set as 500 ± 50r/min.
4. sucking the liquid in each hole, and each hole is filled using the washing buffer (400 μ L) after dilution and is washed, Then washing buffer is sucked completely, repeats the process three times, is washed four times altogether.It must be completely removed washing in each step Otherwise buffer will affect the accuracy of result.After last time is washed, any remaining washing buffer is removed by suction Liquid.Overturning orifice plate sucks residual liquid on clean paper handkerchief.
5. 200 μ L human cell factor conjugates are added into each hole.Adhesive tape is covered again.It is set as at room temperature in revolving speed It is incubated for 2 hours in the horizontal rail microplate oscillator of 500 ± 50r/min.
6. as step 4. in repeat aspiration/washing four times.
7. 200 μ L substrate solutions are added in every hole.It is being protected from light incubation 30 minutes at room temperature.
8. 50 μ L terminate liquids are added in every hole.Color in hole should become yellow from blue.If the color in hole is green Or color change is uneven, can beat plate gently to ensure to be thoroughly mixed.
9. absorbance measurement: using the microplate reader for being set as 450nm, the absorbance in each hole is determined in 30 minutes (optical density, OD) value.Tuning wavelength is set as 540nm or 570nm.According to preset standard concentration and measurement Standard concentration draw standard curve, further according to the OD value of standard curve and sample to be tested calculate sample to be tested in cell factor Concentration.
The cell factor load efficiency of table 2.CSO/H nano-particle modified more micropore cytoskeletons
Group* 2μg/mg 1μg/mg 500ng/mg 250ng/mg
SDF-1EE (%) 89.54±1.39 94.37±1.48 94.41±1.37 93.53±1.87
BMP-2EE (%) 82.73±2.95 92.67±1.82 92.85±1.52 93.78±1.25
* every mg nano particle loads 2 μ g, 1 μ g, 500ng and 250ng cell factor respectively
The cell factor load efficiency of nano-particle modified more micropore cytoskeletons as the result is shown of table 2.Nano particle More micropore cytoskeletons SDF-1 and BMP-2 load efficiency with higher of modification (more than 80%).The physiology of cell factor Concentration is far below 100ng/ml.Therefore, cell factor load capacity is sufficient for the needs of organizational project.
The In-vitro release curves for loading the cell factor (SDF-1 and BMP-2) in nano-particle modified bracket are shown in In Fig. 4.The nano-particle modified bracket of SDF-1 with load shows typical initial burst in preceding 3 detection times point Property release.It shows to mitigate release mode after 5 days.In contrast, the release profiles of BMP-2 are shown similar to medicine for power Zero-order release profile in, whole releasing trend are more stable.However, being mounted with the nano-particle modified of SDF-1 and BMP-2 More micropore cytoskeletons effectively realize the sustained release more than 15 days.Cell factor is carried on nano-particle modified mostly micro- After the cytoskeleton of hole, the bioactivity of the cell factor of sustained release can be kept for a long time.

Claims (9)

1. a kind of more micropore cell scaffold materials, which is characterized in that be made of chitosan, gelatin and agarose, the gelatin-shell Glycan-agar sugared content is 4~5wt%, and the mass ratio of the chitosan, gelatin and agarose is 1:1:(2~3).
2. more micropore cell scaffold materials according to claim 1, which is characterized in that more micropore cell scaffold materials Porosity be 79~82%, swelling ratio be 10.36~15.03, liquid flow rate > 10mlmin-1。
3. the preparation method of more micropore cell scaffold materials as claimed in claim 1 or 2, which is characterized in that by chitosan, gelatin and Agarose is prepared using chemical crosslinking freezing gel method.
4. preparation method according to claim 3, which is characterized in that chitosan be dissolved completely in after acetum be added it is bright Glue mixes well dissolution, and agarose solution is added and mixes, and 5% (v/v) glutaraldehyde solution is added dropwise in blending process It is crosslinked, is subsequently placed at -14 DEG C to be incubated for and prepares within 16 hours freezing gel, thaw at RT prepares more micropore cytoskeletons.
5. a kind of nano-particle modified more micropore cytoskeletons, which is characterized in that by positively charged and can be chemically crosslinked Nano particle be combined into the more micropore cell scaffold materials of gelatine-chitosan-agarose.
6. more micropore cytoskeletons according to claim 5, which is characterized in that the nano particle is chitosan oligosaccharide/heparin Nano particle.
7. the preparation method of the nano-particle modified more micropore cytoskeletons of claim 5 or 6, which is characterized in that by institute It states after nano particle is resuspended in water and obtains nano granule suspension, the more micropore cell scaffold materials of gelatine-chitosan-agarose add Enter in nano granule suspension and shake 6 hours for 37 DEG C, 37 DEG C of reaction 20min of glutaraldehyde solution of 5% (v/v) is added, after cleaning Freeze-drying.
8. preparation method according to claim 7, which is characterized in that the oscillation rate is 60rpm.
9. nano-particle modified more micropore cytoskeletons made from the preparation method of claim 7 or 8.
CN201910800722.8A 2019-08-28 2019-08-28 It is a kind of can be by nano-particle modified more micropore cytoskeletons and preparation method thereof Pending CN110507861A (en)

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CN103239758A (en) * 2012-02-14 2013-08-14 深圳兰度生物材料有限公司 Artificial dermis bracket and preparation method of artificial dermis bracket
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CN102886076A (en) * 2012-09-27 2013-01-23 深圳清华大学研究院 Bone repair porous bracket and rapid forming method
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