CN101148657A - Construction for tissue engineering cartilage modified by transforming growth factor beta2 gene - Google Patents
Construction for tissue engineering cartilage modified by transforming growth factor beta2 gene Download PDFInfo
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- CN101148657A CN101148657A CNA2006101529198A CN200610152919A CN101148657A CN 101148657 A CN101148657 A CN 101148657A CN A2006101529198 A CNA2006101529198 A CN A2006101529198A CN 200610152919 A CN200610152919 A CN 200610152919A CN 101148657 A CN101148657 A CN 101148657A
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Abstract
The present invention discloses process of constructing transforming growth factor beta2 gene modified tissue engineering cartilage. The process includes the separation, culturing and proliferation of adult stem cell from human fat, the transfection of type 5 copying defect adenovirus to the adult stem cell from human fat, the compound of the transfected cell and the polyhydroxy acetic acid/calcium alginate composite as the carrier material, and the formation of in vivo tissue engineering cartilage. Experiment shows that it is possible to form in vivo tissue engineering cartilage through inoculating adult stem cell from human fat to the polyhydroxy acetic acid/calcium alginate composite, and this shows the application foreground of gene reinforced tissue engineering in repairing cartilage defect.
Description
Technical field
The present invention relates to medical science and biomedical engineering field, be specifically related to gene enhanced field of tissue engineering technology.
Background technology
Osteoarthritis or wound etc. cause cartilage to lose and the arthralgia that causes is the disabled major reasons of the elderly.Joint cartilage self repair ability is very limited, and the damage back tends to cause the regression of joint cartilage if can not get effective treatment, finally causes osteoarthritis, symptoms such as pain, ankylosis and dysfunction occur.Methods of treatment commonly used comprises polishing under the arthroscope, boring, little fracture, of the same race from body or allosome cartilage transplantation, from body periosteum or perichondrium transplanting and prosthetic replacement operation etc., yet these traditional treatment schemes often can not generate hyaline cartilage, can't reach the mechanics requirement of joint cartilage bearing load, the prosthetic replacement operation can solve pain and dysfunction problem preferably, but its life-span is limited, is not suitable between twenty and fifty patient.
The rise of organizational project subject provides new selection for the joint cartilage reparation, and its ultimate principle is that the cartilage group tissue that makes new advances of the cell binding matrix material construction of cultured and amplified in vitro is for transplanting.Obtaining of seed cell is the basis that tissue engineering bone/cartilage makes up, the most frequently used seed cell has chondrocyte, mescenchymal stem cell (mesenchymal stem cells at present, MSCs), adipose-derived adult stem cell (adiposederived adult stem cells, ADSCs), embryonic stem cell (embronic stem cells, ESCs).The chondrocyte separates and cultivates comparatively simply, but that the chondrocyte originates is limited, and the vitro culture metabolism is slow, and amplification in vitro is prone to anti-differentiation, and phenotype (particularly II Collagen Type VI) is difficult to keep; The normal needs that obtain of BMSCs carry out local anaesthesia or epidural anesthesia, and it is limited to originate, and the stem cell number is less relatively.Embryonic stem cell has full differentiation potential, can infinite multiplication and be divided into that whole body has 200 surplus kind of cell type, thereby be expected to form any tissue or the organ of body, but control ESCs cell is very complicated to the specific cells differentiation, and the use of ESCs relates to biological safety and ethics problem.ADSCs has demonstrated application prospects because of it is easy to obtain, multiplication capacity strong and have multinomial differentiation potential, becomes hot research in recent years.
Many cytokines to chondrocyte's propagation metabolism and stem cell to playing a significant role aspect chondrocyte's differentiation, as transforming growth factor-beta (TGF-β), Delicious peptide (BMPs), rhIGF-1 (IGFs), fibroblast growth factor (FGFs), but the external application cytokine costs an arm and a leg, and very easily loss in use, gene enhanced Method of Tissue Engineering demonstrates advantage in this respect, be about to goal gene and be transferred to target cell by suitable carriers, and express therein, thereby reach the effect that promotes cell proliferation or differentiation.The endogenous protein of transfectional cell synthesis secretion is through suitable posttranslational modification process, has how discernible part, can be more effectively with the cell surface receptor combination, therefore, its expression product activity is higher, required product amount is littler, has significantly reduced the side effect that exogenous recombinant protein heavy dose is used repeatedly.
Being applied at present that three-dimensional stent material in the cartilage tissue engineered structure can be divided into generally with collagen is the natural materials of representative and with polyglycolic acid (polyglycolic acid, PGA), poly(lactic acid) (poly-L-lactic acid, PLLA) and poly(lactic acid)-polyglycolic acid mixture (PLGA) be the synthetic material of representative, wherein every kind of material all has its superiority-inferiority, the compound of differing materials can be integrated the advantage of various materials, PLGA can make mixture keep certain mechanical strength and specified shape in the PLGA/ Protanal TXF 200 mixture, calcium alginate gel then can be avoided a large amount of losses of cell in the tissue engineering bone/cartilage building process and promote that stem cell breaks up to the chondrocyte, so has demonstrated its superiority in the structure of PLGA/ Protanal TXF 200 mixture in cartilage tissue engineered.
Summary of the invention
The present invention is in order to solve above-mentioned shortcoming, provides a kind of construct in vitro method of organization engineered cartilage of the transforming growth factor beta2 genetic modification based on adipose-derived adult stem cell, in the hope of being applied to the articular cartilage damage reparation.
For achieving the above object, the present invention has taked following technical scheme:
A kind of construction process of organization engineered cartilage of transforming growth factor beta2 genetic modification is provided in a first aspect of the present invention, the separation and Culture and amplification, gene transfection, cell and the carrier that comprise seed cell are compound, and in-vivo tissue through engineering approaches cartilage generates, step is as follows: the separation and Culture of the adult stem cell that (1) is adipose-derived and amplification: get the operation patients subcutaneous lipids, the PBS thorough washing, scissors shreds tissue block; Vibration digestion 45min among the 0.1% collagenase I, add the DMEM that contains 10%FBS and end digestion, the centrifugal 10min of 1200g, the cell mass that obtains suspends the back strainer filtering to remove impurity with nutrient solution, cell inoculation is in culturing bottle, place 370C, 5%CO2 incubator to cultivate, change liquid after two days and remove not adherent red corpuscle.Change liquid weekly twice later on.To cell reach 90% merge after, the exhaustion substratum, with PBS flushing 2 times, add 0.05% trypsinase, place 370C, 5%C02 incubator to hatch 5 minutes, add complete DMEM substratum with in and tryptic activity, blow and beat gently with suction pipe and to make into single cell suspension, by the cultivation of going down to posterity in 1: 2; (2) carry the adipose-derived adult stem cell of replication defective 5 type Adenovirus Transfection of transforming growth factor beta2 gene; (3) transfectional cell and solid support material is compound: with poly(lactic acid)-polyglycolic acid/Protanal TXF 200 mixture as the cell carrier material.Twenty four hours after the transfection, monolayer cell is made single cell suspension after with trysinization, discard nutrient solution after centrifugal, cell is resuspended in 1.2% solution of sodium alginate with 2 * 107/ml concentration, 100 μ l cell suspensions drip gently on poly(lactic acid)-polyglycolic acid mixture, after treating that cell suspension immerses fully, poly(lactic acid)-polyglycolic acid mixture is dipped in the 102mM calcium chloride solution gently, place 370C, 5%CO2 incubator to hatch and take out the cell material mixture after 10 minutes, add after physiological saline washes three times gently and contain serum DMEM substratum.
Preferable, the seed cell in the construction process of above-mentioned organization engineered cartilage is adipose-derived adult stem cell.
Preferable, the adipose-derived adult stem cell in the construction process of above-mentioned organization engineered cartilage is the 3-5 passage cell in generation.
Preferable, the adipose-derived adult stem cell of replication defective 5 type Adenovirus Transfection of transforming growth factor beta2 gene is carried in the construction process utilization of above-mentioned organization engineered cartilage.
Preferable, the solid support material in the construction process of above-mentioned organization engineered cartilage is poly(lactic acid)-polyglycolic acid/Protanal TXF 200 mixture, and wherein the ratio of poly(lactic acid) and polyglycolic acid is 90: 10, and pore size is 100-300 μ m, and porosity is 90%.
Preferable, poly(lactic acid) in the construction process of above-mentioned organization engineered cartilage-polyglycolic acid mixture adopts following method to carry out pre-treatment: 25KGY60Co radiation exposure 12 hours, before using with 75% alcohol-pickled 30 minutes, eliminate surface tension, nutrient solution with serum-free rinses out remaining alcohol repeatedly then, blot with sterile gauze at last, place 24 orifice plates.
Preferable, the compound of cell in the construction process of above-mentioned organization engineered cartilage and material realized by the following method: carry the adipose-derived adult stem cell of 5 type replication defective adenoviral transfections of transforming growth factor beta2 gene after 24 hours, monolayer cell is made single cell suspension with trysinization, discard nutrient solution after centrifugal, cell is resuspended in 1.2% solution of sodium alginate with 2 * 107/ml concentration, cell suspension drips gently on poly(lactic acid)-polyglycolic acid mixture, after treating that cell suspension immerses fully, poly(lactic acid)-polyglycolic acid mixture is dipped in the 102mM calcium chloride solution gently, place 370C, hatched in the 5%CO2 incubator 10 minutes, and made sodiun alginate and calcium chloride fully aggregate into gelatinous Protanal TXF 200.Took out the cell material mixture in 10 minutes, and added after physiological saline washes three times gently and contain serum DMEM substratum.
The method that cartilage generates in a second aspect of the present invention provides a kind of body, the damaged position of hyaline cartilage will implant according to the cell material mixture that above-mentioned organization engineered cartilage construction process obtains.
Owing to taked technique scheme, the present invention to have following advantage: the adipose-derived adult stem cell convenient sources of people, multiplication capacity be strong, have multinomial differentiation potential, little to the donor wound.PLGA can make mixture keep certain mechanical strength and specified shape in the solid support material PLGA/ Protanal TXF 200 mixture, and calcium alginate gel then can be avoided a large amount of losses of cell in the tissue engineering bone/cartilage building process and promote that stem cell breaks up to the chondrocyte.The adipose-derived adult stem cell of transgenic human can implant, shorten cell immediately and obtain and perform the operation time between implanting after being inoculated in solid support material.
Description of drawings
Fig. 1 Ad5-hTGF-β
2The hADSCs material composite of transfection is in the subcutaneous formation bright in color of nude mice class cartilaginous tissue piece, left figure show its volume transplant before no considerable change, right figure demonstration control group graft has dwindling in various degree before than art, appearance color is dark and gloomy.
Fig. 2 experimental group HE dyeing showed cell is embedded in the middle of the lacuna, is surrounded with basophilic cell matrix around the left figure demonstration, and right figure demonstration control group HE dyes and shows to become in a large number the intrusion of fibrous tissue and blood vessel, does not have obvious cartilage generating structure.
Matrix is that obvious blue is different dyes around Fig. 3 experimental group AB-PAS method dyeing showed cell, and left figure points out cell can secrete a large amount of acidic mucopolysaccharides, and right figure points out II Collagen Type VI immunohistochemical staining to be positive.
Embodiment
Below in conjunction with specific embodiments and the drawings, further set forth the present invention.These embodiment and accompanying drawing only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, condition described in " molecular cloning experiment guide " (chopsticks such as the third edition [U.S.] Sa nurse Brooker in 2002, Science Press), or the condition of advising according to manufacturer.
Embodiment, based on the construct in vitro of the organization engineered cartilage of the transforming growth factor beta2 genetic modification of adipose-derived adult stem cell
(1) adipose-derived adult stem cell (human adipose derived adult stem cell, hADSCs) separation and Culture and amplification: a small amount of subcutaneous lipids (the patient male sex of the operation patients that takes a morsel behind the informed consent, 56 years old age, old Hip fracture patient, the row replacement of total hip), PBS washing three times, remove the naked eyes visible vessels, PBS purge three times, scissors shreds fat lump as far as possible is placed in the 50ml centrifuge tube, add 5 times of volume 0.1%I Collagen Type VI enzyme vibration digestion 45min, the back adds the DMEM that contains 10%FBS and ends digestion, and the centrifugal 10min of 1200g removes supernatant and fat and drips, the cell mass that obtains suspends back filter screen (100 μ m) filtration to remove impurity with nutrient solution, and cell inoculation is in 75cm
2Culturing bottle places 37 ℃, 5%CO
2Incubator is cultivated, and changes liquid after two days and removes not adherent red corpuscle.Change liquid weekly later on twice (high sugared DMEM contains 10%FBS).To cell reach 90% merge after, the exhaustion substratum with PBS flushing 2 times, adds 0.05% trypsinase, places 37 ℃, 5%CO
2Hatched in the incubator 5 minutes, add complete DMEM substratum with in and tryptic activity, blow and beat gently with suction pipe and to make into single cell suspension, by the cultivation of going down to posterity in 1: 2.
(2) carry 5 type replication defective adenoviral (Ad5-hTGF-β of transforming growth factor beta2 gene
2) transfection hADSCs: previous experiments should find the suitable transfection plural number that is suitable for the hADSCs transfection, concrete grammar is an amount of cell of inoculation in 24 orifice plates, cell density is with about 90% during to transfection, add serum-free medium 200 μ l in 24 orifice plates, select suitable transfection plural number (MOI=100: 1,200: 1,500: 1,1000: 1), add 5 type replication defective adenoviral (Ad5-EGFP) postoperative infections 90 minutes carry enhanced green fluorescence gene gene, rocked nutrient solution gently once in per 15 minutes, mixing, inhale after 90 minutes and remove original fluid, add fresh complete culture solution, fluorescent microscope is observed transfection efficiency down after 24 hours, as MOI=500: adenovirus can reach 82.5% to the transfection efficiency of hADSCs in the time of 1, and pair cell does not cause obvious damage, and therefore, we select 500: 1 for being suitable for the MOI value of hADSCs.Preceding 24 hours of transfection, an amount of hADSCs of inoculation in the culture dish, cell density changes liquid with about 90% during to transfection before the transfection, adds an amount of serum-free DMEM, and with MOI=500: 1 adds Ad5-hTGF-β
2, infection time is 90 minutes, rocks nutrient solution gently once in per 15 minutes, mixing is inhaled after 90 minutes and is removed original fluid, adds fresh complete culture solution.
(3) preparation of solid support material and transfectional cell and material is compound: poly(lactic acid)-polyglycolic acid mixture (PLGA, poly(lactic acid) wherein: polyglycolic acid is 90: 10, porosity is 90%, and the aperture is 100-300 μ m) entrust Shandong Province medical macromolecular materials key lab to prepare.PLGA accomplishes 6 * 4 * 0.4cm size, 25KGY with blade
60The sterilization in 12 hours of Co radiation exposure is standby.PLGA with 75% alcohol immersion 30 minutes, inhales and removes 75% ethanol in centrifuge tube before using, and with the DMEM purge that does not contain serum three times, places DMEM immersion 2 hours then.Twenty four hours after the transfection, monolayer cell are made single cell suspension after with trysinization, discard nutrient solution after centrifugal, and cell is with 2 * 10
7/ ml concentration is resuspended in 1.2% solution of sodium alginate, and 100 μ l cell suspensions drip gently on PLGA, treat that cell suspension immerses porous PLGA fully after, PLGA is dipped in the 102mM calcium chloride solution gently, place 37 ℃, 5%CO
2Hatched in the incubator 10 minutes, and made sodiun alginate and calcium chloride fully aggregate into gelatinous Protanal TXF 200.Took out the cell material mixture in 10 minutes, and added after physiological saline washes three times gently and contain serum DMEM substratum.The cell of Ad5-EGFP cells transfected and untransfected is operated equally to compare.
(4) body is implanted into: the BALB/C nude mice that selects 4-6 week is for making subcutaneous planting model, and operating process is as follows: abdominal injection hits the Patients Under Ketamine Anesthesia animal under the aseptic condition, will be subcutaneous in 24 hours cell material mixture of vitro culture implantation nude mice, sew up the incision.6 the subcutaneous implantation in nude mice back Ad5-hTGF-β wherein
2The hADSCs material composite of transfection, other 6 hADSCs material composites of implanting the Ad5-EGFP transfection.Draw materials after 3 months, row histology and immunohistochemistry detect.
Draw materials after (5) 3 months and can find β through Ad5-hTGF-
2The hADSCs material composite of transfection is in the subcutaneous formation bright in color of nude mice class cartilaginous tissue piece, no considerable change before its volume is transplanted, HE dyeing showed cell is embedded in the middle of the lacuna, be surrounded with basophilic cell matrix on every side, matrix is that obvious blue is different dyes around the AB-PAS method dyeing showed cell, the prompting cell can be secreted a large amount of acidic mucopolysaccharides, and II Collagen Type VI immunohistochemical staining is positive.And the control group graft has dwindling in various degree before than art, and appearance color is dark and gloomy, and HE dyeing shows and becomes fibrous tissue and blood vessel to invade in a large number.Experimental result prompting Ad5-hTGF-β
2The hADSCs of transfection can generate tissue engineering bone/cartilage in conjunction with PLGA/ Protanal TXF 200 mixture in body, shown that gene strengthens the application prospect of organizational project in cartilage defect is repaired.
Claims (8)
1. the construction process of the organization engineered cartilage of a transforming growth factor beta2 genetic modification, the separation and Culture and amplification, gene transfection, cell and the carrier that comprise seed cell are compound, and in-vivo tissue through engineering approaches cartilage generates, it is characterized in that: the separation and Culture of the adult stem cell that (1) is adipose-derived and amplification: get the operation patients subcutaneous lipids, the PBS thorough washing, scissors shreds tissue block; Vibration digestion 45min among the 0.1% collagenase I adds the DMEM that contains 10%FBS and ends digestion, the centrifugal 10min of 1200g, and the cell mass that obtains suspends the back strainer filtering to remove impurity with nutrient solution, and cell inoculation places 37 ℃, 5%CO in culturing bottle
2Incubator is cultivated, and changes liquid after two days and removes not adherent red corpuscle.Change liquid weekly twice later on.To cell reach 90% merge after, the exhaustion substratum with PBS flushing 2 times, adds 0.05% trypsinase, places 37 ℃, 5%CO
2Hatched in the incubator 5 minutes, add complete DMEM substratum with in and tryptic activity, blow and beat gently with suction pipe and to make into single cell suspension, by the cultivation of going down to posterity in 1: 2; (2) carry the adipose-derived adult stem cell of replication defective 5 type Adenovirus Transfection of transforming growth factor beta2 gene; (3) transfectional cell and solid support material is compound: with poly(lactic acid)-polyglycolic acid/Protanal TXF 200 mixture as the cell carrier material.Twenty four hours after the transfection, monolayer cell are made single cell suspension after with trysinization, discard nutrient solution after centrifugal, and cell is with 2 * 10
7/ ml concentration is resuspended in 1.2% solution of sodium alginate, 100 μ l cell suspensions drip gently on poly(lactic acid)-polyglycolic acid mixture, after treating that cell suspension immerses fully, poly(lactic acid)-polyglycolic acid mixture is dipped in the 102mM calcium chloride solution gently, places 37 ℃, 5%CO
2Hatch in the incubator and take out the cell material mixture after 10 minutes, add after physiological saline washes three times gently and contain serum DMEM substratum.
2. the construction process of the organization engineered cartilage of the transforming growth factor beta2 genetic modification described in the claim 1 is characterized in that seed cell is adipose-derived adult stem cell.
3. the construction process of the organization engineered cartilage of the transforming growth factor beta2 genetic modification described in the claim 1 is characterized in that adipose-derived adult stem cell is the 3-5 passage cell in generation.
4. the construction process of the organization engineered cartilage of the transforming growth factor beta2 genetic modification described in the claim 1 is characterized in that utilizing the adipose-derived adult stem cell of replication defective 5 type Adenovirus Transfection that carries transforming growth factor beta2 gene.
5. the construction process of the organization engineered cartilage of the transforming growth factor beta2 genetic modification described in the claim 1, it is characterized in that solid support material is poly(lactic acid)-polyglycolic acid/Protanal TXF 200 mixture, wherein the ratio of poly(lactic acid) and polyglycolic acid is 90: 10, pore size is 100-300 μ m, and porosity is 90%.
6. the construction process of the organization engineered cartilage of the transforming growth factor beta2 genetic modification described in the claim 1 is characterized in that poly(lactic acid)-polyglycolic acid mixture adopts following method to carry out pre-treatment: 25KGY
60Co radiation exposure 12 hours with 75% alcohol-pickled 30 minutes, is eliminated surface tension before using, and rinses out the alcohol of remnants then repeatedly with the nutrient solution of serum-free, blots with sterile gauze at last, places 24 orifice plates.
7. the construction process of the organization engineered cartilage of the transforming growth factor beta2 genetic modification described in the claim 1, it is characterized in that the compound of cell and material realize by the following method: carry the adipose-derived adult stem cell of 5 type replication defective adenoviral transfections of transforming growth factor beta2 gene after 24 hours, monolayer cell is made single cell suspension with trysinization, discard nutrient solution after centrifugal, cell is with 2 * 10
7/ ml concentration is resuspended in 1.2% solution of sodium alginate, cell suspension drips gently on poly(lactic acid)-polyglycolic acid mixture, after treating that cell suspension immerses fully, poly(lactic acid)-polyglycolic acid mixture is dipped in the 102mM calcium chloride solution gently, places 37 ℃, 5%CO
2Hatched in the incubator 10 minutes, and made sodiun alginate and calcium chloride fully aggregate into gelatinous Protanal TXF 200.Took out the cell material mixture in 10 minutes, and added after physiological saline washes three times gently and contain serum DMEM substratum.
8. the method that cartilage generates in the body is characterized in that the cell material mixture that organization engineered cartilage construction process according to claim 1 the obtains damaged position of hyaline cartilage that implants.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106264776A (en) * | 2016-10-26 | 2017-01-04 | 王东 | Fat milk chylification method |
| CN110124107A (en) * | 2019-04-23 | 2019-08-16 | 哈尔滨医科大学 | A kind of PLGA cytoskeleton and its preparation method and application for articular cartilage reparation |
| CN110885791A (en) * | 2019-12-23 | 2020-03-17 | 上海交通大学医学院附属第九人民医院 | Cartilage regeneration method |
-
2006
- 2006-09-20 CN CNA2006101529198A patent/CN101148657A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106264776A (en) * | 2016-10-26 | 2017-01-04 | 王东 | Fat milk chylification method |
| CN110124107A (en) * | 2019-04-23 | 2019-08-16 | 哈尔滨医科大学 | A kind of PLGA cytoskeleton and its preparation method and application for articular cartilage reparation |
| CN110124107B (en) * | 2019-04-23 | 2021-07-09 | 哈尔滨医科大学 | PLGA cell scaffold for articular cartilage repair and preparation method and application thereof |
| CN110885791A (en) * | 2019-12-23 | 2020-03-17 | 上海交通大学医学院附属第九人民医院 | Cartilage regeneration method |
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