CN106309493A - Cartilage stem cell preparation, and preparation method and application thereof - Google Patents
Cartilage stem cell preparation, and preparation method and application thereof Download PDFInfo
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- CN106309493A CN106309493A CN201610881467.0A CN201610881467A CN106309493A CN 106309493 A CN106309493 A CN 106309493A CN 201610881467 A CN201610881467 A CN 201610881467A CN 106309493 A CN106309493 A CN 106309493A
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- stem cell
- preparation
- cartilage
- cartilage stem
- cell preparation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/16—Blood plasma; Blood serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/19—Platelets; Megacaryocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
Abstract
The invention relates to the technical field of a cell preparation and especially relates to a cartilage stem cell preparation, and a preparation method and application thereof. The cartilage stem cell preparation comprises cartilage stem cells and platelet-rich plasma. The cartilage stem cell preparation provided by the invention can effectively repair joint cartilage injury and has an effect better than the therapeutic effect of sodium hyaluronate combined with mesenchymal stem cells.
Description
Technical field
The present invention relates to cell preparation technical field, particularly to a kind of cartilage stem cell preparation and preparation method thereof and should
With.
Background technology
Articular cartilage is a kind of viscoelastic materials, load-bearer properties that different moving part performances are different.Articular cartilage
This key property and to reduce the effect of articular surface friction be cartilage ultrastructure composition and the basic training of chemical component
Can embody, articular cartilage is formed by sparse cell arrangement, without blood vessel, lymph and neural supply.Articular cartilage is at joint motion
In play an important role, its structure is very fine, science, to adapt to different function needs.Wound or athletic injury often result in pass
The acute injury of joint cartilage, causes arthralgia, swelling and dysfunction, has a strong impact on the quality of life of patient.
At present, the Therapeutic Method of articular cartilage damage specifically includes that
1, joint debridement and flushing: clear up and rinse the arthritis without local symptom the most long-term under arthroscope
Curative effect, is only carefully screening Operation indication, could obtain certain curative effect after arthroscope cleaning.
2, osteochondral fracture is fixed: first removed, inactive downright bad fragment to clinic with throwing cutter, file or curet
The osteochondrosis fragment of upper instability, if with sufficiently large subchondral bone on X-ray sheet, can be fixed.Fixing material
Material: metal hollow nail without a head and bioabsorbable material.
3., bone marrow stimulating technology:
(1) joint polishing plasty: polishing plasty is a kind of traditional Arthroscopic management, utilizes throwing cutter and abrasive drilling to take
Go out the hardening sclerotin of 1～2mm, be driven into euangiotic subcartilaginous osseous lamella, so can promote the formation of fibrin clot, after
Become fibrous cartilage.
(2) micro-fracture: micro-Fracture Technique repairing articular cartilage defect is one of focus of Orthopedic Clinical and experimentation,
It is to be developed by Drilling under cartilage, belongs to bone marrow stimulating technology.
4, cartilage transplantation technology
In sum, complexity and the structural high degree of specificity of normal articular cartilage determines articular cartilage and once damages
It is difficult to after hindering or losing repair.Realize repair of cartilage also by engineering rack at present, but method exists histocompatibility, and
And it is difficult to control the degradation speed of support.
Along with the development of biotechnology, stem cell research in treating various disease models is the most, owing to it has
There is self renewal and polytype cell can be divided into, having a wide range of applications, the most also by soft as treatment joint
The main study subject of bone injury.But directly by stem cell injection treat articular cartilage damage exist cell survival rate on the low side,
The shortcoming of cell proliferation difficulty.Therefore, it is badly in need of providing a kind of pharmaceutical preparation that can effectively treat articular cartilage damage.
Summary of the invention
In view of this, the invention provides a kind of cartilage stem cell preparation and its preparation method and application.This cartilage is dry thin
Born of the same parents' preparation can effectively repairing articular cartilage damage.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides a kind of cartilage stem cell preparation, including cartilage stem cell and platelet rich plasma (PRP).
PRP discharges the somatomedin of multiple high concentration, and its ratio is consistent with internal normal rates, has optimal collaborative work
With.The biological effect of existing monofactor, has again the interaction between various somatomedin.Platelet in PRP has rush
Enter the effect of blood coagulation, cartilage tissue regeneration can be stimulated, promote wound healing.In cartilage stem cell preparation of the present invention, PRP can be obvious
The death of minimizing injection mescenchymal stem cell, and promote propagation and the matrix secretion of cartilage stem cell, without having influence on albumen
Polysaccharide and the type of collagen.Result of study shows that knee articular cartilage defect can effectively be repaired by cartilage stem cell preparation of the present invention.
As preferably, cartilage stem cell concentration in cartilage stem cell preparation is (1～6) × 107Cell/mL.
In the embodiment that the present invention provides, cartilage stem cell concentration in cartilage stem cell preparation is 1 × 107Carefully
Born of the same parents/mL.
In another embodiment that the present invention provides, cartilage stem cell concentration in cartilage stem cell preparation is 3 × 107
Cell/mL.
In another embodiment that the present invention provides, cartilage stem cell concentration in cartilage stem cell preparation is 6 × 107
Cell/mL.
As preferably, the pH value of cartilage stem cell preparation is 6.5～7.5.
As preferably, the osmotic pressure of cartilage stem cell preparation is that 280～320 millis ooze equivalents per liter.
As preferably, cartilage stem cell is autologous cartilage stem cell.
As preferably, platelet rich plasma is autologous platelet rich plasma.
Present invention also offers the application in the medicine of joint injury is repaired in preparation of this cartilage stem cell preparation.
In the embodiment that the present invention provides, joint is knee joint.
As preferably, in the site of administration of medicine is articular cavity.
Present invention also offers the preparation method of this cartilage stem cell preparation, be resuspended in Fu Xue little including by cartilage stem cell
In plate blood plasma, obtain cartilage stem cell preparation.
In the embodiment that the present invention provides, the preparation method of cartilage stem cell is: shredded by cartilaginous tissue, uses pancreas egg
White enzyme digests, and then uses II Collagenase Type to digest, obtains individual cells；Individual cells through original cuiture, pass on
After cultivation, obtain cartilage stem cell.
As preferably, the temperature using trypsin to carry out digesting is 37 DEG C, and the time is 1～2h.
As preferably, the temperature using II Collagenase Type to carry out digesting is 37 DEG C, and the time is 17～20h.
In the embodiment that the present invention provides, the preparation method of platelet rich plasma is: be centrifuged by peripheral blood 400g
10min, is divided into 3 layers；Take upper strata and middle level liquid 400g is centrifuged 5min, be divided into 3 layers；Take upper strata and middle level liquid 1200g is centrifuged
5min, obtains platelet rich plasma.
The invention provides a kind of cartilage stem cell preparation and its preparation method and application.This cartilage stem cell preparation includes
Cartilage stem cell and platelet rich plasma.The device have the advantages that for:
1, the cartilage stem cell preparation that the present invention provides can effectively damage by repairing articular cartilage, and effect is better than hyaluronate sodium
The therapeutic effect of associating mesenchymal stem cells MSCs；
2, as preferably, cartilage stem cell or PRP that the present invention uses are entirely self, and nontoxic, non-immunogenicity is right
Patient damages little, there is not immune rejection problems and pathophorous danger；
3, as preferably, in the site of administration of medicine is articular cavity, articular cartilage is repaired all in articular cavity, is possible to prevent
Cartilage stem cell and hematoblastic loss so that it is secrete somatomedin for a long time in local, keep higher somatomedin concentration
Chondrocyte is conducive to go back to the nest.
Detailed description of the invention
The invention discloses a kind of cartilage stem cell preparation and its preparation method and application, those skilled in the art can borrow
Mirror present disclosure, is suitably modified technological parameter and realizes.Special needs to be pointed out is, all similar replacements and change are to this area
Being apparent from for technical staff, they are considered as being included in the present invention.Method and the application of the present invention have been passed through
Preferred embodiment is described, and related personnel substantially can be to described herein in without departing from present invention, spirit and scope
Methods and applications be modified or suitably change and combine, realize and apply the technology of the present invention.
Biomaterial used, reagent and instrument in cartilage stem cell preparation that the present invention provides and its preparation method and application
All can be buied by market.
Below in conjunction with embodiment, the present invention it is expanded on further:
The separation of embodiment 1 cartilage stem cell and cultivation
1. separate non-stress position articular cartilage under aseptic condition, peel off fascia and the perichondrium of parcel cartilaginous tissue, put
Enter to fill in the culture dish of PBS.
2. the cartilaginous tissue of isolated is shredded into the piece of tissue of 0.3-0.5mm, moves into T25 culture bottle, with containing dual anti-
PBS rinse cartilage 3 times.
3. add cartilage volume 19-15 times 0.25% trypsin, 37 DEG C digestion 1-2 hour after terminate digest.
4. adding 0.02%II Collagenase Type, 37 DEG C digest 17-20 hour.
5. observe under inverted microscope, when, after the individual cells that dissociates, adding DMEM/F12 complete medium by II type
Collagenase dilution terminates digestion.
Filtering with 200 mesh filter screens after 6. cell mass is blown and beaten into individual cells, collect filtrate, 1500rpm/min is centrifuged
5min。
7. abandoning supernatant, addition DS culture fluid is resuspended, 0.2% Trypan Blue, and living cell rate is more than 90%, cell suspension
It is diluted to (2～3) × 105Cell/ml, is inoculated in culture bottle and puts into CO2Incubator is cultivated.
8. original cuiture: by the cell suspension of digestion gained with (2～3) × 105Cell/ml is inoculated in T25 culture bottle,
Every bottle adds 5ml cell suspension.Culture bottle is placed in CO2Incubator is cultivated (37 DEG C, saturated humidity, 5%CO2, 95% is empty
Gas).48h backsight cell attachment situation changes culture fluid, within later every two days, changes culture fluid 1 time.
9. Secondary Culture: observing chondrocyte degree of converging when inverted microscope and reach 90%, incline culture fluid, adds 0.25%
Trypsin 2ml, standing at room temperature 5～10min, if observing that cell shrinkage becomes round under inverted microscope, respective cells is opened
Beginning swims, and now should be rapidly added fresh medium and terminate digestion, and piping and druming cell makes it disperse to form single chondrocyte suspension,
1500rpm/min is centrifuged 5min, cultivates or frozen with standby according to preceding method sub-bottle.
The preparation of embodiment 2 PRP
1. aseptic collection is obtained from external week whole blood 10ml；
2. proceeding to 15ml centrifuge tube, 400g is centrifuged 10min, is now divided into 3 layers, upper strata platelet-poor plasma, middle level richness blood
Platelet, lower floor's hemocyte；
3. transfer upper strata and whole middle level are to new 15ml centrifuge tube, and 400g is centrifuged 5min, is now divided into 3 layers, and upper strata is weary
Thrombocyte plasma, middle level rich platelet, there is a small amount of hemocyte in lower floor；
4. upper strata and middle level being transferred to new 15ml centrifuge tube, tries not to be drawn onto hemocyte, 1200g is centrifuged 5min,
Leave and take the blood plasma needing volume, and resuspended bottom platelet, obtain PRP.
The preparation of embodiment 3 ejection preparation
PRP that cartilage stem cell embodiment 1 obtained obtains through embodiment 2 is resuspended is 1 × 107Cell/ml, is used for noting
It is mapped to affected part.The pH of this cell preparation is 7.2；Osmolarity ranges is that 303 millis ooze between equivalents per liter.
The preparation of embodiment 4 ejection preparation
PRP that cartilage stem cell embodiment 1 obtained obtains through embodiment 2 is resuspended is 3 × 107Cell/ml, is used for noting
It is mapped to affected part.The pH of this cell preparation is 7.3；Osmotic pressure is that 290 millis ooze equivalents per liter.
The preparation of embodiment 5 ejection preparation
PRP that cartilage stem cell embodiment 1 obtained obtains through embodiment 2 is resuspended is 6 × 107Cell/ml, is used for noting
It is mapped to affected part.The pH of this cell preparation is 7.4；Osmotic pressure is that 315 millis ooze between equivalents per liter.
Embodiment 6 knee articular cartilage defect Therapy study
The ejection preparation using the embodiment of the present invention 3～5 to prepare carries out the Therapy study of knee articular cartilage defect.Concrete examination
Proved recipe method is as follows:
Take healthy DABAI piglet 25, be randomly divided into 5 groups:
Experimental group 1: use embodiment 3 ejection preparation to treat；
Experimental group 2: use embodiment 4 ejection preparation to treat；
Experimental group 3: use embodiment 5 ejection preparation to treat；
Positive controls: mesenchymal stem cells MSCs+hyaluronate sodium group
Negative control group: normal saline group.
Artificially manufactured the round speckle damage of a diameter of 5mm by double knee joint, set up knee articular cartilage defect model.Modeling
After, experimental group joint epicoele injects 0.5ml of the present invention, and positive controls joint epicoele is injected and filled between 1% hyaluronate sodium+bone marrow
Matter stem cell 0.5ml, negative control group joint epicoele injecting normal saline 0.5ml, one week 1 time, complete treatment for continuous 8 weeks.Control
Treatment puts to death animal after terminating, and carries out pathological observation, according to Mankin osteoarthritis histologic classification method, comments cartilage
Point, record integration.
Positive controls stem cell medicine preparation method: use the 1% resuspended mesenchymal stem cells MSCs of hyaluronic acid, cell
Concentration is 1 × 107Cell/ml, pH are 7.35, and osmotic pressure is that 295 millis ooze equivalents per liter.
The pH of negative control normal saline is 6.9, and osmotic pressure is that 312 millis ooze equivalents per liter.
Each group pig knee cartilage pathology integration see table:
The table 1 pig knee cartilage pathology table of integrals
| Animal group | Pathology integration |
| Experimental group 1 | 5.16±0.37 |
| Experimental group 2 | 4.72±0.56 |
| Experimental group 3 | 3.34±0.78 |
| Positive controls | 7.12±0.61 |
| Negative control group | 12.58±0.97 |
As can be seen from the above table, experimental group and positive controls all can significantly improve pig knee articular cartilage defect, and experiment
Organize relatively positive controls and also have significant difference, illustrate that ejection preparation of the present invention relatively transparent matter acid sodium associating medulla mesenchyma is dry thin
Born of the same parents' therapeutic effect is notable.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a cartilage stem cell preparation, it is characterised in that include cartilage stem cell and platelet rich plasma.
Cartilage stem cell preparation the most according to claim 1, it is characterised in that described cartilage stem cell is done at described cartilage
Concentration in cell preparation is (1～6) × 107Cell/mL.
Cartilage stem cell preparation the most according to claim 1 and 2, it is characterised in that the pH of described cartilage stem cell preparation
Value is 6.5～7.5.
Cartilage stem cell preparation the most according to any one of claim 1 to 3, it is characterised in that described cartilage stem cell
The osmotic pressure of preparation is that 280～320 millis ooze equivalents per liter.
Cartilage stem cell preparation the most according to any one of claim 1 to 4, it is characterised in that described cartilage stem cell
For autologous cartilage stem cell.
Cartilage stem cell preparation the most according to any one of claim 1 to 5, it is characterised in that described rich platelet blood
Slurry is autologous platelet rich plasma.
7. cartilage stem cell preparation answering in the medicine of joint injury is repaired in preparation as according to any one of claim 1 to 6
With.
8. the preparation method of cartilage stem cell preparation as according to any one of claim 1 to 6, it is characterised in that include soft
Bone stem cell is resuspended in platelet rich plasma, obtains cartilage stem cell preparation.
Preparation method the most according to claim 8, it is characterised in that the preparation method of described cartilage stem cell is: by soft
Osseous tissue shreds, and uses trypsin to digest, and then uses II Collagenase Type to digest, obtains individual cells；Described
Individual cells, after original cuiture, Secondary Culture, obtains cartilage stem cell.
Preparation method the most according to claim 8 or claim 9, it is characterised in that the preparation method of described platelet rich plasma
For: peripheral blood 400g is centrifuged 10min, is divided into 3 layers；Take upper strata and middle level liquid 400g is centrifuged 5min, be divided into 3 layers；Take upper strata
It is centrifuged 5min with middle level liquid 1200g, obtains platelet rich plasma.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108324992A (en) * | 2017-01-19 | 2018-07-27 | 国玺干细胞应用技术股份有限公司 | Composition for cartilage repair and method for repairing articular cartilage |
| CN108653329A (en) * | 2018-05-28 | 2018-10-16 | 广东唯泰生物科技有限公司 | It is a kind of to be used to treat drug of osteoarthritis and preparation method thereof |
| CN108653328A (en) * | 2018-03-26 | 2018-10-16 | 杭州市萧山区中医院 | Application of the mesenchyma stem cell combined platelet rich plasma in preparing the drug for promoting the healing of rotator cuff injury tendon bone complex |
| CN109568660A (en) * | 2017-09-29 | 2019-04-05 | 烟台赛泽生物技术有限公司 | A kind of Preparation method and use of tissue collagen reparation homogenate CCFC |
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| WO2014076200A1 (en) * | 2012-11-15 | 2014-05-22 | Biorigen International Sa | Cell culture supplements |
Non-Patent Citations (2)
| Title |
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| 吴俊等: "富血小板血浆复合软骨细胞构建可注射性组织工程化软骨", 《中国组织工程研究》 * |
| 王忠山等: "富血小板血浆复合软骨细胞用于聚氨酯支架成软骨的研究", 《实用口腔医学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108324992A (en) * | 2017-01-19 | 2018-07-27 | 国玺干细胞应用技术股份有限公司 | Composition for cartilage repair and method for repairing articular cartilage |
| CN109568660A (en) * | 2017-09-29 | 2019-04-05 | 烟台赛泽生物技术有限公司 | A kind of Preparation method and use of tissue collagen reparation homogenate CCFC |
| CN108653328A (en) * | 2018-03-26 | 2018-10-16 | 杭州市萧山区中医院 | Application of the mesenchyma stem cell combined platelet rich plasma in preparing the drug for promoting the healing of rotator cuff injury tendon bone complex |
| CN108653329A (en) * | 2018-05-28 | 2018-10-16 | 广东唯泰生物科技有限公司 | It is a kind of to be used to treat drug of osteoarthritis and preparation method thereof |
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Application publication date: 20170111 |