CN1565643A - Tissue engineered cartilage based on bone marrow mesenchymal stem cell - Google Patents
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- CN1565643A CN1565643A CNA031481507A CN03148150A CN1565643A CN 1565643 A CN1565643 A CN 1565643A CN A031481507 A CNA031481507 A CN A031481507A CN 03148150 A CN03148150 A CN 03148150A CN 1565643 A CN1565643 A CN 1565643A
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Abstract
The invention belongs to tissue engineering field. It makes self body mesenchymal stem cell as seed cell and porous biological ceramics as bracket material. Adding proliferating agent and differentiating agent when cultivating the complex in vitro, transplanting and restoring articular cartilage injury after cultivating in vitro for a period of time The invention is characterized in that it constructs tissue engineering cartilage by employing marrow mesenchymal stem cell and porous biological ceramics for restoring articular cartilage injury. It has an preferably clinical practice prospect.
Description
Technical field
The present invention relates to make the field of tissue engineering technology of human organ and tissue, particularly a kind of organization engineered cartilage and the application in articular cartilage defect is repaired thereof.
Background technology
Cartilaginous tissue is a kind of well differentiated end eventually tissue that does not have blood vessel, no lymphatic vessel and impassivity domination, being difficult to self after the damage repairs, if the damage back can not get effective treatment, tend to cause the regression of articular cartilage, finally cause osteoarthritis, symptoms such as pain, ankylosis and dysfunction occur.And existing clinical treatment method all is difficult to repair effectively the cartilage injury as restriction heavy burden, infrared thermotherapy, electromagnetic stimulation and intraarticular injection transforming growth factor bioactie agents such as (TGF-β) etc., can only temporarily slow down symptom, can not promote repair of cartilage effectively and stop cartilage degeneration.And the hyaline cartilage that operation method comprises cartilage scraping under the arthroscope, boring, little fracture, transplants and all can't generate the cartilage mechanical characteristics from body or allosome cartilage flap transplanting etc. from body periosteum or perichondrium, often do not reach the mechanics requirement that cartilage should bearing load, the result is remission in a short time, but the result of long-term observation is still undesirable.The prosthetic replacement operation can solve arthralgia and dysfunction problem preferably, but shortcomings such as its life-span limited (10～15 years), easily loosening sinking are not suitable for cartilage injury between twenty and fifty patient occurred frequently.Since the notion of proposition organizational projects such as Vacanti in 1987, cartilage makes that because of its simple histologic characteristics cartilage tissue engineered development is rapid, and Cao Yilin successfully produces the organization engineered cartilage great milestone on the organizational project development history especially with people's auricle outward appearance at the nude mice back.Drugs approved by FDA in 1997 first can be used for clinical engineered products C articel, shown the huge potential applicability in clinical practice of cartilage tissue engineeredization product.Although this product comes into operation clinical, also obtained short-term clinical effectiveness preferably, drawbacks limit such as it needs two operations, gap periods is long, expense is expensive and long-term effect is uncertain extensive popularization clinically.Easy and simple to handle, the cycle is short, expense is cheap and the method for stable curative effect is the target that scientists is try hard to gain always.Wherein seed cell and timbering material are two big key factors of organizational project.Cartilage tissue engineered seed cell mainly is a chondrocyte, but limited, the in-vitro multiplication ability in position, source in its body, easily lose drawbacks limit such as phenotype its in external a large amount of amplifications, be difficult to reach needed cell quantity.Mesenchymal stem cells MSCs (MSCs) obtains easily, the in-vitro multiplication ability is strong, can stablize and go down to posterity and do not lose its phenotype, externally under specific condition of culture or intravital inherent microenvironment, can be divided into tissues such as bone, cartilage, muscle, fat and nerve, be the research focus of recent cartilage tissue engineered seed cell, the successful report of repairing cartilage defect in many animal bodies is also arranged.Be used for cartilage tissue engineered timbering material and need excellent biological compatibility, controlled degradability and enough mechanical characteristics promote and support the regeneration of newborn cartilage, cartilage tissue engineered rack material now, as collagen, chitosan, polymer etc. exist mechanical strength not enough, the metabolism acid product is built up, shortcomings such as degradation speed is too fast, and inorganic bio, as porous bio-ceramic, be bata-tricalcium phosphate bioceramic (β-TCP) or α-TCP, the reparation that is used for osseous tissue because of its good bone inducing properties and mechanical strength more, but we recent findings β-TCP also has good chondrocyte induction characteristic, no matter be compound chondrocyte or compound MSCs, repairing articular cartilage is damaged well.
Summary of the invention
The objective of the invention is by method of tissue engineering, is seed cell with the autologous bone marrow mesenchymal stem cells, and compound porous bioceramic makes up organization engineered cartilage, repairs human or animal's articular cartilage defect or damage.The present invention is achieved by the following technical solutions:
1) cultivation of seed cell, the amplification and induce: with the aseptic extraction bone marrow of the syringe of pre-heparinization 5～10ml, separate MSCs with Percoll separating medium density, method is in the 10ml centrifuge tube, the 5ml bone marrow fluid is dripped on the Percol separating medium that equal-volume density is 1073g/l gently, centrifugal 20～30 minutes of 3000rpm, draw white at the interface cell ring, with contain the L-DMEM of 10% self blood plasma liquid former be commissioned to train foster, after growing up to, cell colony uses hyclone instead, both solve former being commissioned to train and supported the topic of difficulty, external a large amount of amplifying cells that again can be fast and convenient.
2) timbering material is prepared: with porous bio-ceramic β-TCP or α-TCP.Separate the disk shape of the prefabricated one-tenth all size of porous bio-ceramic, or be prefabricated into other shape, 60gray as required
60The Co irradiation also can be 75% sterilization in alcohol-pickled 10 minutes～1 hour.With serum-free medium or PBS buffer ethanol is cemented out then to improve hydrophilic with preceding usefulness 95% is alcohol-pickled, the sterilization dry gauze blots liquid.
3) structure of organization engineered cartilage
The 8th～15 code name is produced MSCs make 1～2 * 10
7The cell suspension of/ml drips on timbering material gently, and material overturning gently, makes cell distribution as far as possible evenly.Cell one porous ceramics complex is put into incubator, 37 ℃, 5%CO
2, left standstill under the saturated humidity 6～10 hours, cell is attached in the hole of material and surperficial.Add culture fluid again and (contain 10%FBS; 25ng/mlFGF; 10ng/mlTGFB3, the latter two are short multiplication agent and the differentiation agent of MSCs), cultivated 24 hours
4) body is implanted into
It is consistent with the complex size and the shape that make up that surgical exposure damage articular surface, finishing wound surface make it, and carefully complex embedded defective region, and make it and the surrounding tissue driving fit, closes joint capsule, sew up wound.Postoperative 4 all intrinsic articulation brakings.
Beneficial effect
The invention provides MSCs in-vitro separation, amplification and with the compound structure organization engineered cartilage of material, and the method for using this organization engineered cartilage repairing articular cartilage damage, it is characterized in that cell convenient sources, wound are little, the cartilaginous tissue mechanical strength height of structure, integrate with surrounding tissue.
The specific embodiment
The structure of embodiment 1 organization engineered cartilage
1) mesenchymal stem cells MSCs in-vitro separation and cultured method: 6 monthly age body weight 25Kg ± merino, ketamine 0.01g/kg intramuscular anesthesia, the cropping sterilization of posterior superior iliac spine place, with the puncture of the medullo-puncture needle of 18gauge and marrow aspiration liquid to the syringe that contains 0.1ml (3000u/ml) heparin, centrifugal earlier (1000rpm * 5min), sucking-off blood plasma liquid, add isopyknic L-DMEM mixing then, reuse density is that the Percol separating medium 900g * 25 minutes of 1073g/l isolates nucleated cell, wash centrifugal after, with the L-DMEM mixing that contains 10% self blood plasma liquid, piping and druming is even, with 1 * 10
5/ cm
2Density be inoculated in the culture bottle, change liquid after 48 hours, remove not adherent cell, use the culture fluid that contains 10% hyclone again instead after cell colony occurs.
2) the cell in vitro scaffold complex makes up: β-TCP diameter 8mm, height is 4mm, earlier with 25KGY's
60The Co illumination-based disinfection is used 75% alcohol-pickled elimination surface tension then, and the serum-free medium thorough washing is removed residual alcohol, and sterile gauze blots.Cell concentration to 3 * 10 are adjusted in the conventional digestion of MSCs, collection
7/ ml is inoculated on β-TCP gently, places 6 hours in the incubator, adds culture fluid again and (contains 10%FBS; 10ng/ml TGF-β 3 and 25ng/ml FGF), cultivate that body is implanted into after 24 hours.
The application of embodiment 2 organization engineered cartilages in the sheep articular cartilage defect is repaired
1) body is implanted into: open after the anesthesia sterilization and expose the knee joint condylus medialis femoris, is that the Twist Drill Hole of 8mm is to degree of depth 4mm in its district of bearing a heavy burden with external diameter, one big section holostrome articular cartilage defect of artificial manufacturing, implant MSC-β-TCP complex respectively as experimental group, simple β-TCP repairs and damaged vacant group in contrast.Implant and damaged kitchen range are combined closely, the surface with face the face continuous formation mutually, wound is closed in stitching, brakes heavy burden activity gradually after 2 weeks.
2) result: drew materials in 12 weeks to find to test and organize the existing tangible translucent newborn cartilaginous tissue of defective region to generate, histology result shows as β-TCP and absorbs obviously, surrounded by cartilaginous tissue, the MSCs that implants is converted into a large amount of cartilage matrix of chondrocyte justacrine fully, begins to occur a large amount of blood vessel tissues with the subchondral bone intersection.Defect repair district's surfacing is smooth during 24 weeks, there is transparent cartilaginous tissue to generate, not obvious with normal articular cartilage boundary on every side, histological examination showed β-TCP basic absorption is complete, subchondral bone is organized also and is repaired fully, and newborn cartilaginous tissue is connected closely with cartilage on every side on it, and the cartilage epimatrix is enriched, II Collagen Type VI immunohistochemical staining is obviously positive, and the content of GAG reaches 80% of normal articular cartilage.And simple material group and blank group all do not have reparation fully, small amount of fibers tissue and fibrous cartilage occur.Experimental result prompting MSCs has crucial effects in the process of repairing the cartilage defect of big section holostrome, and β-TCP has also played the effect of supporting MSCs to be divided into chondrocyte within it and attachment point being provided for excretory substrate, position in its absorption is in time filled by cartilage matrix or enchondral ossification, thereby has illustrated that also β-TCP can be used as a kind of ideal timbering material of repairing osteochondral tissue.
Claims (9)
1 one kinds of organization engineered cartilages based on mesenchymal stem cells MSCs.It is characterized in that forming by mesenchymal stem cells MSCs (MSCs) and the inorganic compound structure of porous bio-ceramic.
Mesenchymal stem cells MSCs described in 2 claim 1 is a seed cell, and it is characterized in that can be from from body.This cell have conveniently obtain, the in-vitro multiplication ability is strong, be difficult for the forfeiture phenotype and under external specified conditions or in the body under the inherent microenvironment to the characteristics of mescenchymal tissues such as bone, cartilage, tendon and fat differentiation.
Timbering material described in 3 claim 1 is a porous bio-ceramic, can be bata-tricalcium phosphate (β-TCP), also can be type alpha tricalcium phosphate (α-TCP), it is characterized in that having good biocompatibility, degradation speed is controlled, the mechanical strength advantages of higher, can be prefabricated according to damaged actual size, position and shape.
The in-vitro separation of mesenchymal stem cells MSCs and cultivation realize by the following method in 4 claim 2: at first isolate the plasma fraction in the bone marrow fluid, utilize bioactive ingredients wherein to promote the former generation of MSC to breed.Isolate nucleated cell with the method for density gradient centrifugation then, plant and change liquid removal non-adherent cell after 24 hours, help the purification of MSC.
Use hyclone after cell colony grows up to instead, both solved former being commissioned to train and supported the problem of difficulty, again can external quickly and easily a large amount of amplifying cells.
Porous bio-ceramic described in 5 claim 3 adopts following method to carry out pretreatment: 25KGY
60Co roentgenization 12 hours, compound before earlier with 75% alcohol-pickled 10 minutes, eliminate surface tension, rinse out the ethanol of remnants then repeatedly with the culture fluid of serum-free, blot with sterile gauze at last, be put in 6 orifice plates.
Compound described in 6 claim 1 is to adopt following method to realize: with the mesenchymal stem cells MSCs cultivated with 3 * 10
7The cell density of/ml is planted on β-TCP timbering material, placed 6 hours in 37 ℃, 5% CO2 incubator so that cell and timbering material tight adhesion, add again the culture fluid cultivation after 24 hours body be implanted into.
Culture medium described in 7 claim 6 is commercially available DMEM, it is characterized in that adding 10%FBS, 25ng/ml FGF, 10ng/mlTGF-β 3, and wherein FGF is the multiplication agent of MSCs, and TGF-β 3 is differentiation agents of MSCs).
8 claim 4 kind of described autologous bone marrow plasma fraction removes and contains the different protein ingredient of kind of function surplus in the of 200, comprise multienzyme system (complement system, blood coagulation system, fibrinolytic system and kininogen system), immunoglobulins, protease inhibitor class, traversin class, class lipoprotein, and one group and hemoglobin is transferred and metabolism proteins associated matter class etc., also comprise bioactie agents such as TGF-β, FGF, they have the effect that promotes MSCs propagation and differentiation.
The described organization engineered cartilage of 9 claim 1 can be used for the repairing articular cartilage damage.It is characterized in that cambium has good chondrocyte degree of differentiation, newborn cartilage has suitable mechanical strength, the favorable tissue compatibility, and realize the physiological integration with normal surrounding tissue.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031481507A CN1565643A (en) | 2003-07-03 | 2003-07-03 | Tissue engineered cartilage based on bone marrow mesenchymal stem cell |
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| CNA031481507A CN1565643A (en) | 2003-07-03 | 2003-07-03 | Tissue engineered cartilage based on bone marrow mesenchymal stem cell |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102477419A (en) * | 2010-11-26 | 2012-05-30 | 上海交通大学医学院附属第九人民医院 | Method for in-vitro fusion of stem cells and porous biological material |
| CN103255098A (en) * | 2006-10-23 | 2013-08-21 | 人类起源公司 | Methods and compositions for treatment of bone defects with placental cell populations |
| CN103881971A (en) * | 2012-12-21 | 2014-06-25 | 曾因明 | Culture medium and culture method for culturing and/or amplifying mesenchymal stem cells |
| CN106075585A (en) * | 2016-07-08 | 2016-11-09 | 福建师范大学 | A kind of preparation method based on artificial tendon scaffold materials microstructure through engineering approaches artificial tendon graft |
| CN109152864A (en) * | 2015-12-30 | 2019-01-04 | 艾克斯赛尔治疗公司 | A method of being used to prepare three-dimensional cartilage organoid block |
| CN111870741A (en) * | 2020-08-06 | 2020-11-03 | 厦门大学附属中山医院 | Application of morroniside combined stem cells in preparation of cartilage repair material |
-
2003
- 2003-07-03 CN CNA031481507A patent/CN1565643A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103255098A (en) * | 2006-10-23 | 2013-08-21 | 人类起源公司 | Methods and compositions for treatment of bone defects with placental cell populations |
| CN102477419A (en) * | 2010-11-26 | 2012-05-30 | 上海交通大学医学院附属第九人民医院 | Method for in-vitro fusion of stem cells and porous biological material |
| CN102477419B (en) * | 2010-11-26 | 2014-04-23 | 上海交通大学医学院附属第九人民医院 | Method for in-vitro fusion of stem cells and porous biomaterial |
| CN103881971A (en) * | 2012-12-21 | 2014-06-25 | 曾因明 | Culture medium and culture method for culturing and/or amplifying mesenchymal stem cells |
| CN103881971B (en) * | 2012-12-21 | 2017-02-08 | 曾因明 | Culture medium for culturing and/or amplifying mesenchymal stem cells and culture method thereof |
| CN109152864A (en) * | 2015-12-30 | 2019-01-04 | 艾克斯赛尔治疗公司 | A method of being used to prepare three-dimensional cartilage organoid block |
| CN109152864B (en) * | 2015-12-30 | 2021-12-10 | 艾克斯赛尔治疗公司 | Method for preparing three-dimensional cartilage organoid block |
| CN106075585A (en) * | 2016-07-08 | 2016-11-09 | 福建师范大学 | A kind of preparation method based on artificial tendon scaffold materials microstructure through engineering approaches artificial tendon graft |
| CN106075585B (en) * | 2016-07-08 | 2019-06-21 | 福建师范大学 | A kind of preparation method of tissue-engineered artificial tendon graft based on artificial tendon scaffold material |
| CN111870741A (en) * | 2020-08-06 | 2020-11-03 | 厦门大学附属中山医院 | Application of morroniside combined stem cells in preparation of cartilage repair material |
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