CN105013009B - A kind of preparation method for the timbering material rebuild suitable for Bladder repair - Google Patents
A kind of preparation method for the timbering material rebuild suitable for Bladder repair Download PDFInfo
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- CN105013009B CN105013009B CN201510409396.XA CN201510409396A CN105013009B CN 105013009 B CN105013009 B CN 105013009B CN 201510409396 A CN201510409396 A CN 201510409396A CN 105013009 B CN105013009 B CN 105013009B
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Abstract
The invention discloses a kind of preparation method for the timbering material rebuild suitable for Bladder repair, including prepare glucan chitosan VEGF microballoons；The microballoon of preparation is resuspended, Nano bacteria film is immersed by different gradients in the microspheres solution for the different pore size being resuspended, drying process after standing.The timbering material for being applied to Bladder repair and rebuilding that technical scheme finally to be successfully prepared can release VEGF by sequencing during repairing bladder, vascularization process after promoting in composite time implanting to human body body, improve the success rate that Bladder repair is rebuild.
Description
Technical field
The invention belongs to field of medical technology, is related in a kind of Bladder Tissue Engineering reconstruction research field and is implanted into class life
Thing timbering material construction method, more particularly to a kind of preparation method for the timbering material rebuild suitable for Bladder repair.
Background technology
The bladder disease caused by a variety of causes is always a problem in Urology Surgery clinical treatment.It is although more
Kind autologous tissue has obtained preferable effect, but Comparatively speaking organizational project skill as alternative materials in Bladder repair reconstruction
The inherent shortcoming of art no the former " sacrifice normal structure is cost, with operation wound repair tissue defect ", thus more can conduct
The Main way of following urethra reconstruction development.In existing domestic and foreign literature report for various tissue engineering materials or material-
Research of the seed cell complex in Bladder repair reconstruction is gradually expanded from laboratory to clinic.Wherein being no lack of has small range
The case of actual bladder disease clinical treatment, and obtain satisfying preliminary efficacy.
But being gradually improved relative to seed cell research, which kind of support is selected in Bladder Tissue Engineering reconstruction
Material is ideal but to fail to reach common understanding all the time.At present mainly two major class supports are used in the Tissue Engineering Study of bladder：
Acellular tissue matrices (acellulartissuematrices, ATM) and degradable high polymer material
(syntheticpolymers, SP) two major class.The former can not comply fully with wing at physicochemical property (porosity, pore size etc.)
What Guang was rebuild asks, and extremely difficult quantization regulation；The latter lacks cell recognition signal, is unfavorable for cell recognition and sticks, and degrades
When the sour environment that is formed and its catabolite can also cause the aseptic inflammatory reaction of surrounding tissue
Based on above-mentioned background, at present in the research field, the structure of bacteria cellulose progress associated biomolecule timbering material is utilized
Build and just paid close attention to by increasing researcher.In existing report, the report of biological support structure is carried out using bacteria cellulose
There has been successful story in road.But after the bacterial cellulose stent of various different spaces conformations is implanted in vivo, due to machine
Body lacks corresponding chemical constituent or zymoprotein class so that β-Isosorbide-5-Nitrae glycosidic bond can not be interrupted in bacteria cellulose, thus can not
Voluntarily it is degraded to small molecule glucose.The indwelling of long-time bacterial cellulose stent in may then increase in vivo crystal attachment, calculus
The possibility of formation, foreign body rejection etc., and growth of the normal surrounding tissue along rack surface is limited, this is repaiied with Bladder Tissue Engineering
Multiple physiological processes are not very consistent.Therefore, it is that guarantee is newly-built that promotion organization engineering bladder builds up new blood supply as far as possible in early days, quickly
One of length section Bladder Tissue Engineering postoperative an important factor for reaching ideal effect.Thus planted using the material of preparation
Also need to carry out it further modification processing before the compound and individual Hui Zhi of daughter cell.It is more in conventional research report to prop up
Frame material is mutually tied with angiogenic growth factor (VEGF) or blood vessel hair tonic relevant cell (endothelial cell, endothelial progenitor cells)
Close, to promote the vascularization of timbering material, the length after ensureing in the propagation of seed cell thereon, or even composite time implant
Phase survives.But the operation of such method is relative complex, cost is higher, is not suitable for the modification of extensive tis-sue material, thus away from
From in terms of clinical practice it is also very long with a distance from.Only exogenous VEGF albumen is added can not be effective and lasting in transplanting environment
Ground promotees its vascularization.Seed cell is carried VEGF genes using technique for gene engineering, pass through cell continuous release vegf protein
Promote the vascularization of ischemic tissue, but the method poor controllability, and the risk of virus infection be present.
The content of the invention
In view of this, the invention provides a kind of preparation method for the timbering material rebuild suitable for Bladder repair.
To reach above-mentioned purpose, concrete technical scheme is as follows：
A kind of preparation method for the timbering material rebuild suitable for Bladder repair, comprises the following steps：
Step 1, glucan-chitosan-VEGF sustained-release micro-spheres are prepared；
Step 2, the microballoon of preparation is resuspended, Nano bacteria film is immersed the different pore size being resuspended by different gradients
Microspheres solution in, drying process after standing.
Preferably, by n μ l1~10mg/ml rhVEGF168 solution and m μ l1%w/v glucan water in the step 1
Solution stirs, wherein n：M=1:8~15；1~2.2ml0.1%w/v chitosan solution is progressively added dropwise in above-mentioned solution, and
Stirring, the stirring of 60~95 μ l zinc sulfate is eventually adding, is incubated using 5% mannitol, is centrifuged and be resuspended.
Preferably, the n in the step 1：M=1:10, chitosan solution 1.6ml.
Preferably, 80 μ l1-10mg/ml rhVEGF168 solution is pressed with 800 μ l glucan aqueous solutions in the step 1
700rpm rotating speeds stir 30min.
Preferably, 5min is stirred after adding chitosan solution in the step 1.
Preferably, 80 μ l zinc sulfate stirring 30min is added in the step 1.
Preferably, 30min is incubated using 5% mannitol in the step 1.
Preferably, after in the step 1 by acid-base value regulation and frozen dried, by microballoon pore size control 260~
320nm。
Preferably, 12 hours are stood under 10 DEG C of environment every time when being immersed in the step 2 by different gradients.
Preferably, after completing gradient impregnation in the step 2,50 DEG C of drying process simultaneously give the illumination-based disinfection of cobalt -60.
Relative to prior art, technical scheme has advantages below：
1, the VEGF microballoons of different proportion are added in of the invention in bacteria cellulose, its result can cause final thin
Fungin support produces the ability of corresponding program mode release VEGF activated proteins；
2, according to the difference of bacteria cellulose and mistake VEGF microballoon component ratios, it can make it that the production VEGF of different support is fast
Degree and total amount are sufficiently regulated and controled, and meet the requirement of Bladder Tissue Engineering reconstruction different phase.
Brief description of the drawings
The accompanying drawing for forming the part of the present invention is used for providing a further understanding of the present invention, schematic reality of the invention
Apply example and its illustrate to be used to explain the present invention, do not form inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 is the VEGF microballoon ESEMs of the embodiment of the present invention；
Fig. 2 is the bacterial cellulose stent-VEGF microsphere composite scanning electron microscope (SEM) photographs of the embodiment of the present invention.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other under the premise of creative work is not made
Embodiment, belong to the scope of protection of the invention.
It should be noted that in the case where not conflicting, the feature in embodiment and embodiment in the present invention can phase
Mutually combination.
Embodiments of the invention are done with specific explaination below with reference to accompanying drawing.
A kind of preparation method of timbering material rebuild suitable for Bladder repair of embodiments of the invention, is obtained by preparing
The VEGF- microcapsule granules and manufactured bacteria cellulose material obtained is effectively compound, delays by pre-set stylized time point
On The Drug Release activity VEGF, surrounding tissue capilary will be effectively facilitated and formed.
The active protection for developing into VEGF and sustained release of microcapsule medicament slow release method provide feasibility solution party
Method, activated protein molecule can be slowly discharged in 3~4 weeks in vitro.The VEGF- particulates prepared and manufactured bacteria cellulose
Material is effectively compound, slowly discharges active VEGF by pre-set stylized time point, micro- by surrounding tissue is effectively facilitated
Vascularization.
It is a primary object of the present invention to provide a kind of method for closing bacteria cellulose bracket material control release VEGF, make
Obtain the bacteria cellulose bracket material for being applied to Bladder repair reconstruction being finally successfully prepared and can embody and discharged according to program
The characteristics of VEGF, so as to be suitable for the needs of practical clinical, the final success rate for improving Bladder repair and rebuilding.
To reach above-mentioned purpose, present invention optimization bacteria cellulose material Mechanics of Machinery intensity, porosity, pore size,
Biological degradability, sustained release VEGF- particulates are then effectively combined, are prepared into bacteria cellulose-VEGF- slow-releasing microcapsule timbering materials.
Through WesternBlot technology for detection, the material can slowly discharge active vegf protein molecule in vitro in 3~4 weeks, therefore, lead to
Cross the present invention cross construct have the autologous compound protein material for putting VEGF functions can fully meet that Bladder Tissue Engineering is repaiied
The physics and related biological characteristic requirements of implantable material in multiple reconstruction.
In an embodiment of the present invention, following steps are preferably included：
Take following technical scheme：
Step 1, glucan-chitosan-VEGF microballoons are prepared
The rhVEGF168 solution (1-10mg/ml) of 80 μ l various concentrations and 800 μ l glucan aqueous solutions (1%w/v) are pressed
700rpm rotating speeds stir 30min.1.6ml chitosan solutions (0.1%w/v) are progressively added dropwise in above-mentioned solution, and stir 5min.
It is eventually adding 80 μ l zinc sulfate stirring 30min.30min is incubated using 5% mannitol, centrifuges and is resuspended, eventually through acid-base value
After regulation and frozen dried, as shown in fig. 1, by microballoon pore size control in 300nm or so.
Step 2, bacteria cellulose-VEGF microballoons composite is built
The VEGF microballoons of above-mentioned preparation are resuspended, and will test the Nano bacteria film obtained by different gradients immersion weight
In the microspheres solution of outstanding different pore size, 12 hours are stood under 10 DEG C of environment every time, after completing gradient impregnation, 50 DEG C dry
It is dry to handle and to give the illumination-based disinfection of cobalt -60 to be detected.
The coherent detection of the bacteria cellulose-VEGF microballoon composites of the embodiment of the present invention：
As shown in Figure 2, morphological observation is carried out to bacteria cellulose-VEGF microballoons composite using ESEM.
In microballoon preparation process, part VEGF uses biotin labeling.Completed when prepared by bacteria cellulose-VEGF microsphere composites
Afterwards, it is placed in PBS, in being released into January to the VEGF samples of biotin labeling using ELISA method to it in PBS
VEGF carries out quantitative analysis.
By analyzing different composite material VEGF release mode, the optimal bacteria cellulose of optimization and VEGF microballoons
With pattern.Above-mentioned optimal bacteria cellulose-VEGF microsphere surfaces are inoculated in using people's endothelial cells, are periodically passed through
WesternBlot technology for detection cell surface phosphorylation VEGF-R2 is with for verifying the VEGF activity that composite discharged.
The specific embodiment of the present invention is described in detail above, but it is intended only as example, it is of the invention and unlimited
It is formed on particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and
Substitute also all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and
Modification, all should be contained within the scope of the invention.
Claims (9)
1. a kind of preparation method for the timbering material rebuild suitable for Bladder repair, it is characterised in that comprise the following steps：
Step 1, glucan-chitosan-VEGF sustained-release micro-spheres are prepared
1~10mg/ml of n μ l rhVEGF168 solution and m μ l 1%w/v glucan aqueous solution are stirred, wherein n：M=1:
8~15；1~2.2ml0.1%w/v chitosan solution is progressively added dropwise in above-mentioned solution, and is stirred, and is eventually adding 60~95 μ
L zinc sulfate stirs, and is incubated using 5% mannitol, centrifuges and be resuspended；
Step 2, the microballoon of preparation is resuspended, Nano bacteria film is immersed the micro- of the different pore size of resuspension by different gradients
In ball solution, drying process after standing.
2. the preparation method for the timbering material rebuild as claimed in claim 1 suitable for Bladder repair, it is characterised in that described
N in step 1：M=1:10, chitosan solution 1.6ml.
3. the preparation method for the timbering material rebuild as claimed in claim 2 suitable for Bladder repair, it is characterised in that described
80 μ l 1-10mg/ml rhVEGF168 solution and 800 μ l glucan aqueous solutions are by 700rpm rotating speeds stirring 30min in step 1.
4. the preparation method for the timbering material rebuild as claimed in claim 3 suitable for Bladder repair, it is characterised in that described
5min is stirred after adding chitosan solution in step 1.
5. the preparation method for the timbering material rebuild as claimed in claim 4 suitable for Bladder repair, it is characterised in that described
80 μ l zinc sulfate stirring 30min is added in step 1.
6. the preparation method for the timbering material rebuild as claimed in claim 5 suitable for Bladder repair, it is characterised in that described
30min is incubated using 5% mannitol in step 1.
7. the preparation method for the timbering material rebuild as claimed in claim 6 suitable for Bladder repair, it is characterised in that described
In step 1 by acid-base value regulation and frozen dried after, by microballoon pore size control in 260~320nm.
8. the preparation method for the timbering material rebuild as claimed in claim 1 suitable for Bladder repair, it is characterised in that described
12 hours are stood when being immersed in step 2 by different gradients under 10 DEG C of environment every time.
9. the preparation method for the timbering material rebuild as claimed in claim 1 suitable for Bladder repair, it is characterised in that described
After completing gradient impregnation in step 2,50 DEG C of drying process simultaneously give the illumination-based disinfection of cobalt -60.
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| CN112773941B (en) * | 2020-12-31 | 2022-09-16 | 东华大学 | Chitosan microsphere-bacterial cellulose composite material and preparation and application thereof |
| CN113398336A (en) * | 2021-05-20 | 2021-09-17 | 四川大学华西第四医院 | Composite scaffold material for bladder repair and preparation method thereof |
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| CN101444641B (en) * | 2008-12-24 | 2012-08-08 | 浙江大学 | Three-dimensional large aperture tissue engineering scaffold based on nano-fibers and application thereof |
| CN102586298A (en) * | 2011-12-31 | 2012-07-18 | 无锡瑞融生物医药有限公司 | Preparation of long-acting human vascular endothelial growth factor 165 |
| US20160175487A1 (en) * | 2013-06-28 | 2016-06-23 | Medprin Regenerative Medical Technologies Co., Ltd. | Tissue repair scaffold and preparation method and purpose thereof |
| CN103751851A (en) * | 2014-01-17 | 2014-04-30 | 东华大学 | Preparation method of inorganic/organic multi-drug controlled release composite nano fiber scaffold |
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