CN1626251A - Tissue engineering transplantation of cartilage cell, and preparation method - Google Patents
Tissue engineering transplantation of cartilage cell, and preparation method Download PDFInfo
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- CN1626251A CN1626251A CN 200310109203 CN200310109203A CN1626251A CN 1626251 A CN1626251 A CN 1626251A CN 200310109203 CN200310109203 CN 200310109203 CN 200310109203 A CN200310109203 A CN 200310109203A CN 1626251 A CN1626251 A CN 1626251A
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Abstract
A histoengineered cartilage for treating the damaged cartilage and body surface depression is composed of the pharmacologically acceptable polyacrylamine aqueogel and the cartilage cells chosen from cultured cartilage cells, embryo stem cells, tissue stem cells, marrow interstitical cells, fibrous cells, etc.
Description
Technical field
The present invention relates to the EMB field, it specifically relates to organization engineered cartilage cell transplantation and preparation method thereof.
Background technology
Osteomalacia is one of clinical common disease, as the various articular surface illness that caused by factors such as wound, infection, autoimmunity, tumors; Auricular cartilage, nose, laryngeal cartilages support damaged, lopsided; And the wound of bone in children epiphyseal growth plate, early close, necrosis etc.Because chondrocyte belongs to the extremely weak terminally differentiated cells of hypertrophy ability, lack regeneration capacity, the post-traumatic reparation of cartilage is difficulty very, so must utilize cartilage or other substitution material to repair.Limited from body cartilage transplantation source, cause easily for the damaged influence in district and supply district's function, thereby be restricted in clinical practice.Cartilaginous tissue is transplanted extensive use once, but finally makes cellular exposure cause immunological rejection among circulating antibody owing to bearing a heavy burden and wearing and tearing, at present clinical few application the to the utmost.Though the biomaterial of synthetic can be replaced cartilage, exist to implant loosening and not anti abrasive shortcoming, and easily cause and infect and get rid of external as allosome.
Along with the appearance of tissue engineering technique, the research of repair of cartilage has obtained very big development.Cartilage is to use this technical research the earliest, also is one of research tissue maximum, with fastest developing speed and that succeed the earliest.Its basic skills is that the cartilaginous tissue cell seeding that normal function is arranged with In vitro culture is on the good cell carrier natural or synthetic that can be degraded by body of biocompatibility, form cell-carrier complexes, Hui Zhi is in body then, to repair cartilage defect, finally reach the purpose of the cartilaginous tissue that is formed with function.Cartilage tissue engineered research of present stage mainly concentrates on following three aspects: 1) chondrocyte Source Study; 2) research of the exploitation of cell carrier material and modification thereof; 3) the organization engineered cartilage tissue construction and the substitution studies of disease being decreased tissue.The modification of the wherein constituent of cell carrier material, surperficial physicochemical property and carrier material and the biological behaviour of moulding and chondrocyte are closely related, directly the form and the function of the cartilaginous tissue that forms become the important content that cartilage tissue engineering rack is studied.
At present many natural and supports synthetic are applied to cartilage tissue engineered, from hydrogel, loose structure to fibre substrate.Synthetic material is divided into two kinds of solid and fluent materials, and solid is representative with poly-hydroxycaproic acid (PGA), polylactic acid (PLA) and both copolymer.Its major advantage shaping has certain intensity, but there still have many problems to have at the aspects such as control of biocompatibility, physicochemical property, degradation rate to be to be solved.Have good biology as poly-hydroxycaproic acid (PGA) and think intermiscibility, can finely induce, promote sticking, breed and breaking up of chondrocyte as cartilage tissue engineered rack material, form cartilaginous tissue, but the PGA degraded is very fast, be prone to and burst apart, PGA integral body is subsided.And, can cause local pH value to descend because the PGA degraded is too fast, and the catabolite hydroxy acid is assembled in the part, chondrocyte is poisoned and even death.The own propylene of fluent material oxidation (Pluronic) is representative, though Pluronic can form cartilage the animal that complete immunologic function is arranged in body, but this gel is water soluble at normal temperatures, thereby can't do any external tissue culture's experiment, the degradation time of material can't be regulated in a big way, and can not be moulding, and whether it finally lapses in vivo and can exist harmful effect it be unclear that to body.Natural material is more with collagen, gelatin and fibrin research, its the most outstanding advantage is a no antigen, biocompatibility is good, with the extracellular matrix structural similarity, participate in the agglutination of tissue, yet, as the chondrocyte support time, often subside in advance and do not reach the purpose that induces cartilaginous tissue because its degraded is too fast.This shows that cytoskeleton has become the major limitation of cartilage tissue engineered clinical application, thereby seek the focus that timbering material has more suitably become cartilage tissue engineered research.
In addition, the cartilaginous tissue cell is originated also very important, health cartilage transplantation commonly used in the past, and it is limited to originate on the one hand, and causes easily for distinguishing damaged or influencing the function for the district, therefore is very limited in clinical practice.Extensive use allosome cartilage transplantation once afterwards, but owing to bear a heavy burden and wearing and tearing finally makes cellular exposure among circulating antibody and cause immunological rejection, uses seldom clinically at present.Therefore, produce chondrocyte In vitro culture, that normal function is arranged, synthetic material good with biocompatibility recited above then, that can be accepted by body forms chondrocyte-carrier complexes, it is the organization engineered cartilage cellular transplant, Hui Zhi is in body again, to repair cartilage defect, finally reach the cartilaginous tissue that is formed with function, this is the present stage direction mainly studied of cartilage through engineering approaches.
Summary of the invention
One of purpose of the present invention is to select a kind ofly have that acceptable pharmaceutically is safe, good stability, preparation method are simple, the more reliable synthesized polymer material of effect is as the carrier material of cell engineering cartilage cell transplantation.
Two of purpose of the present invention is to provide a kind of by In vitro culture, and the chondrocyte and the application in the engineered technology of chondrocyte thereof of normal function are arranged.
Three of purpose of the present invention is the optimum number that contain chondrocyte in the selected cell engineering cartilage cell transplantation.
The present invention implements by following technical scheme
A first aspect of the present invention is to select a kind of pharmaceutically acceptable polyacrylamide hydrophilic gel as carrier material, will be mixed in equably among the carrier at the chondrocyte that In vitro culture has a normal function then, makes it to become cell republicanism cartilage cell.
Many tissues in the organism are similar with hydrogel structure, and bio-tissue is made up of cell and extracellular matrix, and extracellular matrix components is the hydrogel structure that is made of protein, polysaccharide etc., as the vitreous body of eye and cartilage matrix etc.Polyacrylamide hydrophilic gel is a kind of novel soft tissue charges, and when water content can reach more than 97% and to use as carrier, water absorption rate generally was controlled at 1-20 doubly.Facts have proved that polyacrylamide hydrophilic gel has good biocompatibility, it is a kind of novel bulking of soft tissue, can be used for respectively organizing in body surface and the body the damaged soft tissue filling of pitting and the capacity of augmenting tissue, as face, trunk, extremity, etc. the depression of tissue, enlarge the bosom, temporo cheek and penis increase slightly the filling of vesicoureteral reflux etc.Insert material as long term human and have favorable tissue intermiscibility, non-immunogenicity, avirulence, nonirritant, no heat source response, no mutagenesis and lethal mutation effect.Have good chemical stability, insert human body for a long time and do not produce the chemical constitution change, good corrosion resistance does not produce poisonous leachable.The present invention is according to above-mentioned result of study, and the hydrogel that selected polyacrylamide is made is as the carrier of chondrocyte, develops the tissue engineering bone/cartilage cellular transplant of a kind of safe, good stability, remarkably productive, strong operability.
Described chondrocyte is that the former generation to the of In vitro culture is hexabasic from body or allogeneic chondrocyte, also can be through to the inductive embryonic stem cell of chondrocyte table shape, tissue stem cell, bone marrow stromal cell, fibroblast, fat stem cell, epithelial cell, Tenocyte cell, and with the mixture of chondrocyte.
The cultivation of chondrocyte with separate
Cartilage extract part to human body or animal carries out partly sterilised earlier, takes out cartilage, can be articular cartilage, rib or other cartilages.Peel off away cartilage, be cut into 5mm * 5mm cartilage sheet, with phosphate buffer (PBS, contain each 100u/ml of mycillin) flushing 2 times, the 3mg/ml II Collagen Type VI enzyme (Worthington that adds 2 times of chondrify volumes, Freehold, NJ, USA), place digestion 8h in 37 ℃ of constant temperature shaking tables, filter centrifugal with 150 order nylon mesh screens.The cell that is precipitated out is washed secondary with PBS, Ham, the F-12 culture fluid is made cell suspension, contain 10% hyclone in the suspension, L-glutaminate 300Ug/ml, vitamin C 50ug/ml, each 100ug/ml of penicillin and streptomycin, remove supernatant before the use, pair cell is counted reuse trypan blue chromoscopy chondrocyte vigor, and vigor is greater than the structure of 50% available cartilaginous tissue.
Experimental technique (comprising step):
The chondrocyte made is mixed with acceptable polyacrylamide hydrophilic gel pharmaceutically, form graft, wherein said chondrocyte is that In vitro culture former generation to the is hexabasic from body or allogeneic chondrocyte, described polyacrylamide hydrophilic gel is pharmaceutically acceptable, its water absorption rate is controlled at 1-20 doubly, and the best is 2-12 times.
The content of chondrocyte is 1 * 10 in graft
6Individual cell/ml-1 * 10
8Individual cell/ml.
Described chondrocyte is also optional from through to embryonic stem cell, tissue stem cell, bone marrow stromal cell, fibroblast, fat stem cell, epithelial cell, the Tenocyte cell of chondrocyte phenotype induction, and with the cell mixing of chondrocyte.
Make the organization engineered cartilage graft, can be injected directly into the subcutaneous of human body or animal, each position such as body surface recess or cartilage defect place, easy to operate.
Description of drawings
Fig. 1, experimental group implantation place gross examination of skeletal muscle
Fig. 2, different times chondrocyte-polyacrylamide hydrophilic gel form the cardinal principle specimen (2,8,16,24 week) of cartilage
Fig. 3, chondrocyte-polyacrylamide hydrophilic gel are formed by the island cartilaginous tissue, and chondrocyte is embedded in the cartilage lacuna, see to have and are dyed bluish violet, flaky hydrogel (HE * 100,2 weeks).
The island cartilage of Fig. 4, chondrocyte-polyacrylamide hydrophilic gel still exists, see that mature chondrocytes is wrapped in the cartilage lacuna, in fibrous tissue and in the cartilaginous tissue, have and dyed bluish violet, flaky hydrogel, do not have tangible inflammatory cell infiltration (HE * 100,8 weeks).
The island cartilage that Fig. 5,6, chondrocyte-polyacrylamide hydrophilic gel form still exists, and quilt is dyed bluish violet in the cartilaginous tissue, flaky hydrogel still exists, and does not see obvious degradation, do not see yet and degenerate and ossified phenomenon, histologic characteristics and 8 weeks similar (HE * 100,16,24 weeks).
For example practical
One, the separation of chondrocyte:
Long wind hybridized pig: in 8 ages in week, malely do not limit body weight 6-8Kg.Ketamine (2mg/Kg), sodium pentobarbital (0.2ml/Kg) intramuscular anesthesia, after the local geramine tincture sterilization, get bilateral and wide cartilage, peel off perichondrium, be cut into 5 * 5mm size cartilage sheet, phosphate buffer (PBS, contain green grass or young crops, each 100u/ml of streptomycin) flushing both sides time add the 3mg/ml II Collagen Type VI enzyme (Worthington of 2 times of chondrify volumes, Freehold, NJ USA), places digestion 8h in 37 ℃ of constant temperature shaking tables, filter centrifugal with 150 order nylon mesh screens, the cell that is precipitated out is washed 2 times with PBS, Ham, and the F-12 culture fluid is made cell suspension, contain 10% hyclone in the suspension, L-glutaminate 300Ug/ml, vitamin C 50ug/ml, green grass or young crops, each 100ug/ml of streptomycin, cell counting, every pig can gather in the crops 1 * 10 approximately
8Individual chondrocyte, trypan blue chromoscopy chondrocyte vigor, vigor is greater than 50% the structure that can be used as cartilaginous tissue.
Two, chondrocyte-polyacrylamide hydrophilic gel complex preparation:
Chondrocyte suspension in former generation, the first generation, the second filial generation, the third generation, the 4th generation, the 5th generation, the 6th generation is centrifugal respectively, the supernatant that inclines are incited somebody to action wherein monobasic cell precipitation and the abundant mixing of polypropylene amine hydrogel, and making cell concentration is 40 * 10
6/ ml cell-polyacrylamide hydrophilic gel complex, it is standby to draw complex with the 2ml syringe.
Three, chondrocyte-polyacrylamide hydrophilic gel composite body is implanted into and draws materials
Under the aseptic condition, chondrocyte-polyacrylamide hydrophilic gel complex is inoculated in pig under the corium of body abdomen lateral wall with thick needle, every some inoculation 0.3ml, and the injection back is pressed with have gentle hands and is made it to be hemispherical.Observe the injection site and have or not redness, heating, scleroma and soft durometer thereof, and drew materials respectively in the 2nd, 8,16,24 weeks, the engineered tissue of injecting back tissue reaction, toxicity and formation is estimated with the injection back.
Gross examination of skeletal muscle: result such as above-mentioned shown in Figure 1, all do not occur obviously red and swollen around the implantation place skin, quality is softer, as shown in Figure 2, the different time cartilage that forms of drawing materials is creamy white in chondrocyte-polyacrylamide hydrophilic gel composite body, form is irregular disk shape, and certain elasticity is arranged, outward appearance and normal cartilage likeness in form.
Histology: result such as above-mentioned Fig. 3, Fig. 4, Fig. 5 and shown in Figure 6, HE dyeing sees that chondrocyte-polyacrylamide hydrophilic gel has the island cartilaginous tissue of being cut apart by fibrous tissue to form during 2 weeks, the ripe chondrocyte of part is embedded in the cartilage lacuna, in fibrous tissue and in the cartilaginous tissue, have and dyed bluish violet, flaky hydrogel, do not have tangible inflammatory cell infiltration, do not have new vessels yet and grow into.HE dyeing sees that the island cartilage that chondrocyte-polyacrylamide hydrophilic gel forms still exists during 8 weeks, see that mature chondrocytes is wrapped in the cartilage lacuna, in fibrous tissue and in the cartilaginous tissue, have and dyed bluish violet, flaky hydrogel, do not have tangible inflammatory cell infiltration.16, the HE dyeing of 24 whens week sees that the island cartilage that chondrocyte-polyacrylamide hydrophilic gel forms still exists, quilt is dyed bluish violet in the chondrocyte, flaky hydrogel still exists, do not see obvious degradation, do not see yet and degenerate and ossified phenomenon that histologic characteristics is similar with 8 weeks.
Compare prior art, advantage of the present invention and good effect
The material of described organization engineered cartilage graft can be to be injected directly into each position in human body such as subcutaneous, body surface recess or cartilage defect place or the animal, and method is simple.Using polypropylene amide hydrogel has made up a kind of new organization engineered cartilage graft that safety is higher, effect is more reliable, stability is better, operability is stronger as cell carrier.Clinical showing after material of the present invention is transplanted in human body or animal body, in the different time sampling, proves that established cartilage is creamy white, and form is irregular, and certain elasticity is arranged, and outward appearance is similar to normal cartilage with the histocytology feature.Adopt this cartilage tissue engineered technology, can thoroughly avoid using cytotoxicity and the bad phenomenon such as formation cartilage degradation, calcification that other cell carrier materials may cause.
Claims (12)
1, a kind of through engineering approaches cartilage graft, it is characterized in that selecting for use pharmaceutically acceptable polyacrylamide hydrophilic gel is carrier, chondrocyte evenly is compound in makes chondrocyte-carrier complexes in the carrier.
2, organization engineered cartilage graft as claimed in claim 1, the amount that it is characterized in that containing in the described graft carrier chondrocyte is 1 * 10
6Individual cell/ml-1 * 10
8Individual cell/ml.
3, organization engineered cartilage graft as claimed in claim 1, it is characterized in that described polyacrylamide hydrophilic gel is by acrylic acid, methacrylamide polymer, be a kind of be rich in hydrophilic group because of the tridimensional network macromolecular material, its dry weight content is about 2.8%, and the water yield can reach more than 97% after the swelling.Have good biocompatibility, water absorption rate is general in the practical application grasps at 1-20 doubly, and the best is 2-12 times.
4, organization engineered cartilage graft as claimed in claim 1 is characterized in that described chondrocyte is that In vitro culture former generation to the is hexabasic from body or allogeneic chondrocyte.
5, organization engineered cartilage graft as claimed in claim 1, it is characterized in that described chondrocyte also can be to derive from through to embryonic stem cell, histiocyte, bone marrow stromal cell, fibroblast, fat stem cell, epithelial cell, the Tenocyte cell of chondrocyte phenotype induction, and with the cell mixing of chondrocyte.
6, organization engineered cartilage graft as claimed in claim 1 is characterized in that described chondrocytes expressed II Collagen Type VI.
7, organization engineered cartilage graft as claimed in claim 1 is characterized in that can adding in the graft or compound other various cells, somatomedin and various transgene component.To keep chondrocyte growth and substrate synthesis capability.
8, the preparation method of organization engineered cartilage graft as claimed in claim 1, it is characterized in that counting behind the chondrocyte suspension in former generation, the first generation, the second filial generation, the third generation, the 4th generation, the 5th generation, the 6th generation is centrifugal respectively, the supernatant that inclines, generation chondrocyte that then will be wherein mixes with acceptable acrylamide hydrogel pharmaceutically, makes chondrocyte-carrier complexes.
9, the preparation method of organization engineered cartilage graft as claimed in claim 8 is characterized in that the polyacrylamide hydrophilic gel water content is 1-20 a times of original dry state, and containing the chondrocyte amount in the hydrogel is 1 * 10
6Individual cell/ml-1 * 10
8Individual cell/ml.
10, the preparation method of organization engineered cartilage graft as claimed in claim 8, being characterised in that described chondrocyte is In vitro culture former generation to the hexabasicly to obtain the cartilage position from body or allogeneic chondrocyte and be not particularly limited, can be articular cartilage, rib or its elsewhere cartilage.
11, the preparation method of organization engineered cartilage graft as claimed in claim 8, it is characterized in that described chondrocyte also can be derive from through through to the embryonic stem cell of chondrocyte phenotype induction, tissue stem cell, bone marrow stromal cell, fibroblast, adipose cell, epithelial cell, Tenocyte cell and with the mixture of chondrocyte.
12, as claim 8, the preparation method of 9 or 10 described organization engineered cartilage grafts, when it is characterized in that chondrocyte prepares, earlier cartilage being extracted part carries out disinfection, take out cartilage, peel off perichondrium, and be cut into the cartilage sheet of 5 * 5mm size, with phosphate buffer (PBS, contain each 100u/ml of mycillin) wash 2 times, add 3mg/ml II Collagen Type VI enzyme (Worthington, the Freehold of 2 times of chondrify volumes, NJ, USA), place digestion 8h in 37 ℃ of constant temperature shaking tables, filter centrifugal with 150 order nylon mesh screens, the cell that is precipitated out is washed secondary with PBS, Ham, the F-12 culture fluid is made cell suspension, contains 10% hyclone in the suspension, L-glutaminate 300Ug/ml, vitamin C 50ug/ml, green grass or young crops, each 100ug/ml of streptomycin, cell counting, with trypan blue chromoscopy chondrocyte vigor, vigor is greater than 50% the structure that can be used as cartilaginous tissue.
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| CN 200310109203 CN1626251A (en) | 2003-12-09 | 2003-12-09 | Tissue engineering transplantation of cartilage cell, and preparation method |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009003375A1 (en) * | 2007-06-29 | 2009-01-08 | Shanghai Tissue Engineering Life Science Co. Ltd. | Tissue engineering tendon and construction methods in vitro thereof |
| CN102458496A (en) * | 2009-05-15 | 2012-05-16 | 新加坡南洋理工大学 | Composition for manufacturing a scaffold for tissue engineering, and a method of making it |
| CN106421919A (en) * | 2016-11-08 | 2017-02-22 | 华南生物医药研究院 | Composition for cartilage injury repair |
| CN106540326A (en) * | 2016-11-08 | 2017-03-29 | 华南生物医药研究院 | Purposes of the chondrocyte composition in medicine is prepared |
| CN106552288A (en) * | 2016-11-08 | 2017-04-05 | 华南生物医药研究院 | The method for preparing chondrocyte composition |
| CN108690205A (en) * | 2018-05-28 | 2018-10-23 | 深圳市第二人民医院 | II Collagen Type VI of one kind and polyacrylamide composite hydrogel and its preparation and application |
| CN114854675A (en) * | 2021-01-20 | 2022-08-05 | 上海软馨生物科技有限公司 | Method for realizing cartilage regeneration by inoculating gel cartilage to framework structure |
| CN115581811A (en) * | 2022-11-03 | 2023-01-10 | 上海交通大学医学院附属第九人民医院 | Autologous tissue engineering living cell meibomian plate substitute and preparation method and application thereof |
-
2003
- 2003-12-09 CN CN 200310109203 patent/CN1626251A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009003375A1 (en) * | 2007-06-29 | 2009-01-08 | Shanghai Tissue Engineering Life Science Co. Ltd. | Tissue engineering tendon and construction methods in vitro thereof |
| CN102458496A (en) * | 2009-05-15 | 2012-05-16 | 新加坡南洋理工大学 | Composition for manufacturing a scaffold for tissue engineering, and a method of making it |
| CN102458496B (en) * | 2009-05-15 | 2016-06-01 | 新加坡南洋理工大学 | For manufacturing compositions and the production method thereof of scaffold for tissue engineering |
| CN106421919A (en) * | 2016-11-08 | 2017-02-22 | 华南生物医药研究院 | Composition for cartilage injury repair |
| CN106540326A (en) * | 2016-11-08 | 2017-03-29 | 华南生物医药研究院 | Purposes of the chondrocyte composition in medicine is prepared |
| CN106552288A (en) * | 2016-11-08 | 2017-04-05 | 华南生物医药研究院 | The method for preparing chondrocyte composition |
| CN108690205A (en) * | 2018-05-28 | 2018-10-23 | 深圳市第二人民医院 | II Collagen Type VI of one kind and polyacrylamide composite hydrogel and its preparation and application |
| CN114854675A (en) * | 2021-01-20 | 2022-08-05 | 上海软馨生物科技有限公司 | Method for realizing cartilage regeneration by inoculating gel cartilage to framework structure |
| CN115581811A (en) * | 2022-11-03 | 2023-01-10 | 上海交通大学医学院附属第九人民医院 | Autologous tissue engineering living cell meibomian plate substitute and preparation method and application thereof |
| CN115581811B (en) * | 2022-11-03 | 2024-04-02 | 上海交通大学医学院附属第九人民医院 | Autologous tissue engineering living cell meibomian substitute and preparation method and application thereof |
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