CN102416201A - Preparation method and application for transforming growth factor composite scaffold for in-vivo cartilage repair - Google Patents
Preparation method and application for transforming growth factor composite scaffold for in-vivo cartilage repair Download PDFInfo
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- CN102416201A CN102416201A CN2011104142900A CN201110414290A CN102416201A CN 102416201 A CN102416201 A CN 102416201A CN 2011104142900 A CN2011104142900 A CN 2011104142900A CN 201110414290 A CN201110414290 A CN 201110414290A CN 102416201 A CN102416201 A CN 102416201A
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Abstract
The invention provides a preparation method and application of a transforming growth factor composite scaffold for in-vivo cartilage repair. Growth factors are wrapped by chitosan so as to prepare sustained-release microspheres, and the sustained-release microspheres are placed in a chitosan scaffold so as to form the composite scaffold; and the transforming growth factor composite scaffold is mainly used for adsorbing and introducing bone mesenchymal stem cells into cartilage cells in vivo under the action of sustained-release transforming growth factors so as to repair cartilage defect. The invention has the advantages that: the composite scaffold introduces the repair and growth of cartilage in vivo, has dynamic mechanical simulation, can be adhered to and integrated with cartilage tissues of a host easily, and can shape an outline which is matched with that of joints easily, avoid differentiation of cells cultured in vitro and immunological rejection reaction of implanted cells, and reduce pathogen transmission, teratogenesis and dedifferentiation, has a good curative effect when being applied to clinic and has good economic benefit, and scale industrialization can be realized.
Description
Technical field
What the present invention relates to is a kind of method for preparing and application thereof of tissue engineering material; Particularly porous chitosan adsorbs self mesenchymal stem cells MSCs; Induce down at the slow release transforming growth factor, in body, induce application, belong to the tissue engineering material field into chondrocyte.
Background technology
Just adopt the way repairing articular cartilage of boring under the cartilage as far back as nineteen fifty-nine Pridie; Subsequently; There are many people to use penetration under the cartilage in succession,, can recover damaged of destructive joint in a short time like method repairing articular cartilages such as Drilling or little fracture art under wearing and tearing arthroplasty, the cartilage; But say that from long-term effect repair tissue can not bear the mechanical pressure in joint because of fibrocartilaginous formation.At present, make bone repair of cartilage to the normal bone cartilage of damage, still do not have perfect method.The bone cartilage grafting is repaired cartilage defect with the normal hyaline that has the vigor chondrocyte, and the advantage that keeps cartilage biochemistry and bio-mechanical characteristic is arranged, and autotransplantation is better to child's effect, can supply the limited shortcoming of cartilage but exist from body.On the contrary, then there is liable to infection deficiency in heteroplastic transplantation, and long-term effect can not be affirmed.Periosteum and perichondrium grafting are potential methods, can guide graft to grow into cartilage, but exist donor amount finite sum repair tissue to be prone to form the defective of endochondral ossification.
Development along with cell culture technology; The achievement in research of organization engineered cartilage has been lighted the enthusiasm of cartilage defect property diseases such as people's cure osteoarthritis; Especially the application of transgenic technology makes the stem cell or the chondrocyte of the cytokine of people's short cell proliferation of having expected transfection and directed differentiation can continuously form cartilaginous tissue, forms the balance with degraded to reach cartilage.Yet ideal organization engineered cartilage also has a segment distance in that to be applied to also have many problems to have before clinical to be solved from clinical practical application.Though the through engineering approaches cartilage that forms in external abundant cultivation fully is used to repair cartilage defect its advantage is arranged; Promptly can regulate the condition of culture of cell and tissue; Cellular metabolism can receive more better control with differentiation; Yet its shortcoming also is conspicuous, with the integration of host tissue and the secure fixation that reaches on the mechanics all be masty problem.
The NEW TYPE OF COMPOSITE timbering material that comprises TGF-β 1 sustained-release microsphere; In the cartilage defect model of under cartilage, holing; The medullary cell that utilizes self is the damaged ability of repairing articular cartilage in vivo, to explore the damaged practicable Therapeutic Method of repairing articular cartilage.
Summary of the invention
At present, make bone repair of cartilage to the normal bone cartilage of damage, still do not have perfect method.The bone cartilage grafting is repaired cartilage defect with the normal hyaline that has the vigor chondrocyte, and the advantage that keeps cartilage biochemistry and bio-mechanical characteristic is arranged, and autotransplantation is better to child's effect, can supply the limited shortcoming of cartilage but exist from body.On the contrary, then there is liable to infection deficiency in heteroplastic transplantation, and long-term effect can not be affirmed.Periosteum and perichondrium grafting are potential methods, can guide graft to grow into cartilage, but exist donor amount finite sum repair tissue to be prone to form the defective of endochondral ossification.
In order to solve exist in the foregoing technology not enough local, the invention provides a kind of method for preparing that is used for the transforming growth factor compound rest of repair of cartilage in the body, specifically be a kind of method for preparing and application thereof of sustained-release micro-spheres shell compound rest; Promptly utilize chitosan parcel somatomedin; The preparation sustained-release micro-spheres is put into chitosan stent, forms compound rest; Be mainly used in absorption and induce self mesenchymal stem cells MSCs; Induce down at the slow release transforming growth factor, in body, induce application, thereby repair cartilage defect into chondrocyte.
With internal energy cell and the signaling molecule of catching self of compound rest implant, its cell differentiation procedure takes place in vivo with meeting the requirements, and the activity of the formed chondrocyte of long term maintenance.Break up in the physiological environment of repairing that is organized in body interior mechanics load and rebuild.The original position repairing growth more easily and host's cartilaginous tissue adheres to and integration, also can mould the profile with the joint coupling easily.Before the existing many basic problems of through engineering approaches cartilage of above-mentioned relevant In vitro culture do not solve, as if more correspond to reality with this propagation of self cell and the method for differentiation reparation cartilage defect utilized in the body.
Be used to realize that concrete technical scheme of the present invention is following:
The preparation of transforming growth factor sustained-release micro-spheres: adopting emulsion-crosslinking method, is emulsifying agent with Si Ben-80, and liquid paraffin is an oil phase; It is water that chitosan is dissolved in acetic acid, under agitation forms the W/O emulsion, with the sodium tripolyphosphate crosslinking curing; With isopropyl alcohol and distilled water rinsing microsphere repeatedly; Lyophilization gets microsphere, and in microsphere, lyophilizing obtains sustained-release micro-spheres once more with certain density transforming growth factor solution impregnation;
The preparation of compound rest: with finite concentration chitosan acetic acid solution freezing in template, capable again lyophilization promptly obtains the porous chitosan support.Then sustained-release micro-spheres is put into support, form compound rest after the lyophilization.
Acetic acid concentration is 1%-5%, chitosan concentration 1%-5% (w/v), and the concentration of Si Ben-80 is 1%-8%; Oil-water ratio is 1%-10%; The concentration 0.1g/ml of sodium tripolyphosphate, the concentration of transforming growth factor solution is at 5ng/ μ l, the mass ratio 3-25ng/g of transforming growth factor and microsphere.
Transforming growth factor impregnated in the microsphere 24 hours under 4 ℃.The lyophilization temperature is at-60 ℃.
Each compound rest contains transforming growth factor 3-25ng.
Below technical scheme of the present invention is made further description:
The preparation that is used for the transforming growth factor compound rest of repair of cartilage in the body specifically may further comprise the steps:
TGF-β 1 microsphere preparation: the 150mg chitosan is dissolved in the acetic acid of 6ml 2%; Then 30ml liquid paraffin and 1.5ml Si Ben-80 are mixed, under magnetic agitation, slowly dropwise add the chitosan acetic acid solution, be the W/O emulsion; After stirring 1 hour, it is crosslinked with it dropwise to add 100g/LTPP10ml with the 1ml syringe with 20/component velocity; Continue to stir 1 hour, deposition, isopropyl alcohol, distilled water is rinsing repeatedly; Lyophilization got yellow powder powder microsphere in 48 hours; With 1mg chitosan microball and 2.5 μ g/mlTGF-β, 15 μ L, fully soak at 4 ℃ during pH=7.4, expansion 24h;
The preparation of chitosan stent: the chitosan powder is dissolved in 2% acetic acid solution, and stirring is mixed spends the night, and vacuum filtration is removed insoluble impurity, and to obtain final concentration be that the clarification chitosan gel rubber of 20 g/L is subsequent use; With 24 porocyte culture plates is template, injects chitosan gel rubber ,-80 ℃ of cryogenic refrigerator freeze overnight; Freezer dryer freezed 1 hour in advance, in negative 53 degree freezer dryers, continued the about 48h of lyophilizing under the evacuation, to bone dry; The gained support makes its gradient aquation through 100%, 95%, 80%, 70% ethanol, and every gradient respectively is 1h, spends the night with 0.5N NaOH/ alcohol mixed solution (4v/1v) neutralization; Last a large amount of 70% alcohol flushings; It is neutral surveying pH value, and three-dimensional porous rack is processed in lyophilization once more, the thick 10mm of this three-dimensional porous rack; Diameter 15mm, all support ethane via epoxyethane sterilizations are subsequent use
: phase separation method prepares chitosan stent, and the chitosan microball of getting the preparation of an amount of emulsion-crosslinking method ultrasonicly makes its dispersion in pure water; Splash into gradually in the made support; Vacuum filtration does not have bubble, and lyophilization obtains compound rest once more, and ethane via epoxyethane sterilization cold preservation is subsequent use.
Beneficial effect of the present invention is embodied in:
1, the compound rest preparation is simple, and is less demanding to experimental facilities, is convenient to general just popularization.
2, compound rest is induced the repair of cartilage growth in internal in-situ, and having dynamic mechanical stimulates, and the cartilaginous tissue easier and host adheres to and integrates, and also can mould the profile with the joint coupling easily.
3, avoid the variation of cultured cell in vitro performance and the immunological rejection of implantation cell.
4, reduce transmission of pathogen, teratogenesis, dedifferente.
5, be applied to clinical generation good efficacy.
6, industrial scaleization has good economic benefit.
Embodiment below in conjunction with concrete further specifies the present invention
The specific embodiment
Embodiment 1,
A kind of method for preparing that is used for the transforming growth factor compound rest of repair of cartilage in the body, wherein: this method mainly is to utilize chitosan parcel somatomedin, and the preparation sustained-release micro-spheres is put into chitosan stent, forms compound rest.
Embodiment 2,
A kind of method for preparing that is used for the transforming growth factor compound rest of repair of cartilage in the body, wherein:
Emulsion-crosslinking method is adopted in the preparation of transforming growth factor sustained-release micro-spheres, is emulsifying agent with Si Ben-80; Liquid paraffin is an oil phase, and it is water that chitosan is dissolved in acetic acid, under agitation forms the W/O emulsion; With the sodium tripolyphosphate crosslinking curing, with isopropyl alcohol and distilled water rinsing microsphere repeatedly, lyophilization gets microsphere; In microsphere, lyophilizing obtains sustained-release micro-spheres once more with certain density transforming growth factor solution impregnation;
The preparation of compound rest, with finite concentration chitosan acetic acid solution freezing in template, capable again lyophilization promptly obtains the porous chitosan support.Then sustained-release micro-spheres is put into support, form compound rest after the lyophilization.
Embodiment 3,
A kind of method for preparing that is used for the transforming growth factor compound rest of repair of cartilage in the body; Wherein: acetic acid concentration is 1%-5%, chitosan concentration 1%-5% (w/v), and the concentration of Si Ben-80 is 1%-8%; Oil-water ratio is 1%-10%; The concentration 0.1g/ml of sodium tripolyphosphate, the concentration of transforming growth factor solution is at 5ng/ μ l, the mass ratio 3-25ng/g of transforming growth factor and microsphere.All the other are with embodiment 1, embodiment 2.
Embodiment 4,
A kind of method for preparing that is used for the transforming growth factor compound rest of repair of cartilage in the body, wherein: transforming growth factor impregnated in the microsphere 24 hours under 4 ℃, the lyophilization temperature is-60 ℃; Each compound rest contains transforming growth factor 3-25ng.All the other are with embodiment 1, embodiment 2, embodiment 3.
Embodiment 5,
1, animal divides into groups
24 new zealand white rabbits are divided into four groups at random; Cause the holostrome cartilage defect of bilateral femur coaster portion; Implant TGF-β 1 microsphere/chitosan stent (MS-TGF group), TGF-β 1/ chitosan stent (CS-TGF group), simple chitosan stent group (CS group) respectively, other establishes the blank group.Used compound rest ethane via epoxyethane sterilization.
2, operation is implanted
Art two knee joint depilations the previous day, the ketamine intramuscular anesthesia of 44mg/kg according to dosage.With two knee joints is the center, and the routine disinfection drape with center very under the patella, is got two knee joint inner incisions; Be about 3cm, cut skin and subcutaneous tissue, interior lateral incision capsula articularis genus, patella outwards overturns; Fully go down on one's knees, appear the femoral intercondylar cartilage, in the positive middle part of femoral intercondylar, the 4.2mm electric drill is slowly holed; Break through subchondral bone, make holostrome cartilage defect model, use thin Kirschner wire instead and get through pulp cavity, remove the bone bits; See have blood to flow out after, implant different stent materials respectively, gross examination of skeletal muscle sees that all supports all adsorb bone marrow blood Cheng Hongran, the control defects group is spacious puts.Will not wash, direct layering is sewed up the incision, and it is free movable to put back in the cage after animal is clear-headed.Three days penicillin prevention infection of postoperative, otch iodophor disinfection every day secondary.
3, gross examination of skeletal muscle and tissue scoring
Air embolism is put to death animal respectively postoperative January, March, observes articular cavity and has or not and infect or hydrops, and whether total joint form color and luster changes, defective region size, shape, color and luster, quality, and repair tissue is marked with the row Masuoka such as integration of boundary on every side.
4, groupization dyeing is identified
Observation of cell form, substrate metachromasy under capable CD34 of postoperative support in January and the CD44 SABC light microscopic identify whether self mesenchymal stem cells MSCs absorption is arranged.
5, pathological observation and scoring
With the defect is the center, and little electric saw collection comprises the DF bone cartilage piece of normal cartilage and subchondral bone, fixing 48 hours of 4% paraformaldehyde, EDTA decalcification, routine paraffin wax embedding, 5um section, row Toluidine blue staining and II Collagen Type VI immunohistochemical staining.Adopt Wakitani scoring assessment cartilaginous tissue repairing quality.
Conclusion: damaged 4mm is an animal model with the rabbit femoral condyle, gets through pulp cavity, implants compound rest; Adsorb self mesenchymal stem cells MSCs, change at slow release under the inducing of the long-living long factor, gradate chondroblast; The performance biological activity, thus cartilage defect repaired, recover function of joint.
Embodiment 6,
Method as above divides 3ng, 6ng, 12.5ng, four groups of sustained-release micro-spheres shells of 25ng to gather compound rest, draws materials postoperative January, February, March, detects as above, and the result: repair of cartilage is dose dependent, increases with concentration, and the cartilage-derived growth situation is good more.3ng sustained-release micro-spheres shell gathers the compound rest group and draws materials postoperative January, February, March and detect the repair of cartilage degree and be respectively 82%, 86%, 91%; 6ng sustained-release micro-spheres shell gathers the compound rest group and draws materials postoperative January, February, March and detect the repair of cartilage degree and be respectively 88%, 92%, 97%; Draw materials postoperative January, February, March and detect the repair of cartilage degree and be respectively 91%, 94%, 99% 12.5ng the sustained-release micro-spheres shell gathers the compound rest group; 25ng sustained-release micro-spheres shell gathers the compound rest group and draws materials postoperative January, February, March and detect the repair of cartilage degree and be respectively 95%, 98%, 100%.
Claims (5)
1. method for preparing that is used for the transforming growth factor compound rest of repair of cartilage in the body, it is characterized in that: this method mainly is to utilize chitosan parcel somatomedin, and the preparation sustained-release micro-spheres is put into chitosan stent, forms compound rest.
2. a kind of method for preparing that is used for the transforming growth factor compound rest of repair of cartilage in the body as claimed in claim 1 is characterized in that:
Emulsion-crosslinking method is adopted in the preparation of transforming growth factor sustained-release micro-spheres, is emulsifying agent with Si Ben-80; Liquid paraffin is an oil phase, and it is water that chitosan is dissolved in acetic acid, under agitation forms the W/O emulsion; With the sodium tripolyphosphate crosslinking curing, with isopropyl alcohol and distilled water rinsing microsphere repeatedly, lyophilization gets microsphere; In microsphere, lyophilizing obtains sustained-release micro-spheres once more with certain density transforming growth factor solution impregnation;
The preparation of compound rest, with finite concentration chitosan acetic acid solution freezing in template, capable again lyophilization promptly obtains the porous chitosan support; Then sustained-release micro-spheres is put into support, form compound rest after the lyophilization.
3. according to claim 1 or claim 2 a kind of is used for the method for preparing of the transforming growth factor compound rest of repair of cartilage in the body; It is characterized in that: acetic acid concentration is 1%-5%, chitosan concentration 1%-5% (w/v), and the concentration of Si Ben-80 is 1%-8%; Oil-water ratio is 1%-10%; The concentration 0.1g/ml of sodium tripolyphosphate, the concentration of transforming growth factor solution is at 5ng/ μ l, the mass ratio 3-25ng/g of transforming growth factor and microsphere.
4. like claim 1 or 2 or 3 described a kind of method for preparinies that are used for the transforming growth factor compound rest of repair of cartilage in the body, it is characterized in that: transforming growth factor impregnated in the microsphere 24 hours under 4 ℃, the lyophilization temperature is-60 ℃.
5. like claim 1 or 2 or 3 or 4 described a kind of method for preparinies that are used for the transforming growth factor compound rest of repair of cartilage in the body, it is characterized in that: each compound rest contains transforming growth factor 3-25ng.
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| CN106377799A (en) * | 2016-10-13 | 2017-02-08 | 南通大学附属医院 | Preparation method of dental pulp stem cell and chitosan scaffold complex |
| CN107441555A (en) * | 2017-07-27 | 2017-12-08 | 中山大学附属第医院 | A kind of artificial skin and preparation method of controllability release medicine |
| CN109106988A (en) * | 2018-08-15 | 2019-01-01 | 杭州市萧山区中医院 | Astragalus polyose is for promoting the regenerated application of neoplastic skin blood vessel network in organization engineering skin |
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| CN113215089A (en) * | 2020-10-11 | 2021-08-06 | 西北农林科技大学 | Manufacturing method of edible chitosan 3D gel scaffold for cell culture meat |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104056304A (en) * | 2014-07-02 | 2014-09-24 | 昆明医科大学第一附属医院 | Growth-factor-chitosan-microsphere loaded DBM support joint cartilage repairing material |
| CN105435310A (en) * | 2015-12-24 | 2016-03-30 | 钟达 | Method for preliminarily constructing tissue-engineered cartilages in vitro from Wharton jelly of umbilical cord |
| CN106377799A (en) * | 2016-10-13 | 2017-02-08 | 南通大学附属医院 | Preparation method of dental pulp stem cell and chitosan scaffold complex |
| CN107441555A (en) * | 2017-07-27 | 2017-12-08 | 中山大学附属第医院 | A kind of artificial skin and preparation method of controllability release medicine |
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| CN109833509B (en) * | 2019-01-18 | 2021-10-15 | 太阳雨林（厦门）生物医药有限公司 | Multiple sustained-release vascular embolism drug-loading composition |
| CN110448732A (en) * | 2019-08-27 | 2019-11-15 | 中南大学湘雅医院 | Region stem cell induced activity removes cell bone tendon interface page bracket |
| CN110448732B (en) * | 2019-08-27 | 2022-03-29 | 中南大学湘雅医院 | Regional stem cell induced active decellularized bone tendon interface page support |
| CN110801513A (en) * | 2019-12-17 | 2020-02-18 | 河南汇博医疗股份有限公司 | Composite sustained-release particle loaded with growth factor and preparation method thereof |
| CN113215089A (en) * | 2020-10-11 | 2021-08-06 | 西北农林科技大学 | Manufacturing method of edible chitosan 3D gel scaffold for cell culture meat |
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Application publication date: 20120418 |