CN108342356A - A kind of cartilage graft and its construction method - Google Patents
A kind of cartilage graft and its construction method Download PDFInfo
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- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
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- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
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Abstract
The invention discloses a kind of cartilage graft and its construction methods.The cartilage graft is cartilage diaphragm or injectable cartilage graft；The cartilage graft contains cartilage cell's in vitro culture and is formed by cartilage diaphragm or cartilage micro-assembly robot.
Description
Technical field
The present invention relates to medicine and biomedical engineering field, relate more specifically to a kind of cartilage graft and its structure side
Method.
Background technology
Cartilage defect is clinical common disease, is more common in ear caused by congenital reason and wound, nasal cartilages deformity and lacks such as,
Cartilage defect etc. caused by articular trauma and regression.Since cartilaginous tissue self-healing ability is poor, so all kinds of cartilage damages
Reparation be always clinical treatment problem.In recent years, Minimally Invasive Surgery is quickly grown, and injection fillers treatment becomes research hotspot.
Currently used syringeability filler mainly has hyaluronic acid, the degradation materials such as synthetic or animal derived collagen.To the greatest extent
The satisfaction of these injectable materials short run effects is managed, but its curative effect is only capable of maintaining 6-9 months, need to inject repeatedly and there may be serious
Complication, such as chronic granuloma, blood vessel embolism are even blinded and cerebral infarction.
As the rise of tissue engineering technique, especially cartilage cell and the inside and outside regeneration of biodegradable material complex are soft
The success of bone, tissue engineering bone/cartilage provide new approaches for the treatment and injection fillers beauty of cartilage defect.Traditional tissue
Engineered cartilage refers to so that cell is mixed with degradable timbering material by organizational engineering method, forms cell-Material cladding
It after object, is transplanted or is injected into internal privileged site and form cartilaginous tissue, reached reparation cartilage defect or be locally filled with beauty
Moulding purpose.It has many advantages, such as it is easy to operate, safe and effective, minimally invasive or noninvasive, be easier to fill up irregular defect, be cartilage
The important development direction of organizational project application study.But there are still following problems for it：1. low generation cartilage cell quantity is not
Foot:When cartilage cell-material composite regeneration of cartilage, cell concentration require it is very high (>50×106/ml).So using low generation
When cartilage cell, cell quantity cannot be satisfied the demand of regeneration large volume cartilage.And to reach cell quantity requirement, it need to cut big
Volume cartilaginous tissue, donor source is limited and wound is very big.2. high generation cartilage cell easily dedifferentes：Cartilage cell is through external big
After amount amplification, although cell quantity can reach requirement, the height generated loses for cartilage cell it is easy to appear dedifferenting
Lose the ability of regeneration of cartilage.3. moulding difficulty：Cartilage cell-syringeability material composite has runny characteristic, causes
It is moulding more difficult after compound internal injection.4. regenerating bone or cartilage effect is uncontrollable：Due to by cell viability, material degradation rate,
The series of factors such as the inflammatory reaction that individual generates material influence, and cause final regeneration of cartilage quality and volume can not be accurate
Control.Problems above significantly limits extensive use of the regenerating bone or cartilage technology in clinical all kinds of cartilage defect repairs.
Therefore, there is an urgent need in the art to provide regenerating bone or cartilage effect stability, controllable, easy moulding and incidence of complications
Low cartilage graft.
Invention content
The present invention is intended to provide a kind of cartilage graft and its construction method.
In the first aspect of the present invention, a kind of cartilage graft is provided, the cartilage graft contains external regeneration
Cartilage diaphragm or cartilage micro-assembly robot.
In another preferred example, the cartilage graft contains cartilage micro-assembly robot and injectable medium；The cartilage micro-group
It is 10 to knit with the weight ratio of injectable medium:0-5；The injectable medium be selected from physiological saline, phosphate buffer (PBS),
Hyaluronic acid or autoserum.
In another preferred example, the external regeneration cartilage diaphragm or cartilage micro-assembly robot are prepared by following step：
(1) it cartilage cell's cultured and amplified in vitro and passes on；
(2) chondrocyte induction culture is carried out after being inoculated with the cartilage cell of passage, obtains the cartilage diaphragm of external regeneration；
(3) regenerated cartilage diaphragm obtains cartilage micro-assembly robot outside cutting body.
In another preferred example, cartilage cell's inoculum density in step (2) is 3 × 105A cell/cm2-1×107It is a
Cell/cm2；More preferably 1 × 106A cell/cm2-5×106A cell/cm2。
In another preferred example, 1-50ng/ml transforming growth factor is contained in the culture solution of the chondrocyte induction culture
(Transforming growth factor beta 1, TGF β 1), 10-200ng/ml insulin-like growth factor (Insulin
Like growth factor, IGF) and 10-200ng/ml dexamethasone.
More preferably, also one or more containing following substances in the culture solution of the chondrocyte induction culture：1-
50ng/ml insulin, 1-50mg/ml basic fibroblast growth factors (basic fibroblast growth factor,
BFGF), 1-25ng/ml transferrins, 1-20mmol/ml 3-isobutyl-1-methylxanthines (3-Isobutyl-1-
Methylxanthine, IBMX), 1-50ng/ml sodium selenites, 5 × 10-2- 1mg/ml proline, the white egg of 1-100mg/ml serum
In vain, 1-20mg/ml beta -mercaptoethanols, 1-50mg/ml L-Glutamines, 1-20 μ g/ml linoleic acid and 10-200ng/ml dimensions
Raw element C phosphates.
In another preferred example, the cartilage cell is the generation secondary culture of the 1st, 2,3,4,5,6,7 or 8；More preferably
5 generation of 2nd generation-the.
In another preferred example, the cartilage cell is from self or allogeneic；More preferably Auricular cartilage, nose
Interval and costal cartilage.
In the second aspect of the present invention, a kind of preparation side of the cartilage graft provided present invention as described above is provided
Method, the method include step：
(1) cartilage cell is subjected to plane amplification, and is passaged to the generation of the 1st, 2,3,4,5,6,7 or 8；
(2) cartilage cell's high density that the generation of the 1st, 2,3,4,5,6,7 or 8 passes on is seeded on Tissue Culture Dish and is carried out
Chondrocyte induction culture obtains external regeneration cartilage diaphragm；
(3) the outer regeneration of cartilage diaphragm of cutting body forms cartilage micro-assembly robot；
(4) it is obtained after cleaning external regeneration cartilage diaphragm or cartilage micro-assembly robot physiological saline or PBS as described above
Cartilage graft provided by the invention.
In another preferred example, by after cutting cartilage micro-assembly robot physiological saline or after PBS cleans, with physiological saline,
The injectable mediums such as PBS, hyaluronic acid or autoserum are mixed to form injectable cartilage graft.
In the third aspect of the present invention, a kind of cartilage graft provided present invention as described above is provided in structure group
Application in weaver's journey cartilage tissue.
In the fourth aspect of the present invention, provides a kind of cartilage cell and build application in cartilage graft in vitro.
In another preferred example, in vitro during structure cartilage graft by cultured and amplified in vitro and the cartilage of passage
Cell is with 3 × 105A cell/cm2-1×107A cell/cm2Density (more preferably 1 × 106A cell/cm2-5×106It is a
Cell/cm2) be inoculated with after, containing 1-50ng/ml TGF β 1, fill in rice to 10-200ng/ml IGF and 10-200ng/ml
Chondrocyte induction is carried out in the culture solution of pine, makes its regeneration of cartilage diaphragm in vitro；More preferably, also contain in the culture solution and be selected from
Following one or more kinds of substances：1-50ng/ml insulin, 1-50mg/ml bFGF, 1-25ng/ml transferrins, 1-
20mmol/ml IBMX, 1-50ng/ml sodium selenites, 5 × 10-2- 1mg/ml proline, 1-100mg/ml seralbumins, 1-
20mg/ml beta -mercaptoethanols, 1-50mg/ml L-Glutamines, 1-20 μ g/ml linoleic acid and 10-200ng/ml vitamin Cs
Phosphate.
In another preferred example, the cartilage diaphragm of external regeneration is cut to obtain cartilage micro-assembly robot.
Accordingly, the present invention provides regenerating bone or cartilage effect stabilities, controllable, easily moulding and low incidence of complications soft
Bone graft.
Description of the drawings
Fig. 1 shows the preparation process of cartilage graft provided by the invention；Wherein,
A is auricular cartilage；B is cartilage cell's in vitro culture；C is the cartilage diaphragm of external regeneration；D cuts for cartilage diaphragm
The cartilage micro-assembly robot formed afterwards；E, F are cartilage micro-assembly robot and a small amount of physiological saline (about 10:1 ratio) it is mixed to form injectable
Property cartilage graft is injected into the self subcutaneous abdomen of sheep, draws materials after 24w substantially and Histological results；G is the outer structure of cartilage membrane body
Build tubulose cartilage；H is that sheet cartilage is formed in cartilage membrane body；I is that cartilage diaphragm construct inner tissue is engineered tracheae.
Specific implementation mode
For problems of the prior art and difficulty, inventor has found can be first with a small amount of Primary chondrocyte
Large amplification in vitro is realized that cell concentration increases by geometric progression and is wanted substantially to cell quantity with meeting large volume regenerating bone or cartilage
It asks；Then it is special at chondrocyte induction cultivating system to be combined by high density inoculation, reverses high generation cartilage cell to dedifferente existing
As realizing external large volume regenerating bone or cartilage, obtaining cartilage diaphragm.Finally, using cartilage diaphragm or will be obtained by its machine cuts
Cartilage micro-assembly robot prepares cartilage graft.On this basis, the present invention is completed.
Term
Term " cartilage cell " and " chondroblast " may be used interchangeably, and all refer to the conventional separation using this field
Cell technology, what is obtained from self or allosome cartilaginous tissue can secrete II Collagen Type VIs (collagen II), glycosaminoglycan
The polygonal attached cell of the cartilage specificities matrix such as (glycosaminoglycan, GAG).
Term " separation of cartilage cell " refers to the process for separating cartilage cell from tissue.
Term " amplification of cartilage cell " refers to the mistake being largely proliferated in an in vitro environment to obtain a large amount of cartilage cells
Journey.
Term " passage of cartilage cell " refers to the process of that will persistently be passed under cartilage cell in vitro condition of culture.
Term " chondrocyte induction culture solution " refers to having containing specific biochemical composition and promoting chondroblast external regeneration
The culture solution of cartilaginous tissue.
Term " chondrocyte induction culture ", which refers to, provides special biochemical environment, makes to have the cell expression of cartilage differentiation potential soft
Bone phenotype and the process for having into cartilage ability.
Term " cartilage diaphragm " and " external regeneration () cartilage diaphragm " may be used interchangeably, and all refer to that Subchondral drilling is thin
The culture solution that born of the same parents provided through the invention after high density inoculation cultivates the sheet cartilaginous tissue of formation in vitro.
Term " cartilage micro-assembly robot " refers to cartilage diaphragm through machine cuts or shreds the cartilage fragment to be formed.
Term " injectable cartilage graft " and " syringeability cartilage graft " may be used interchangeably, and all refer to that can pass through
The mode of injection is transplanted to the cartilage graft in animal body.
Chondroblast
The source of chondroblast is not particularly limited in the present invention, can be the cartilage cell of human or animal, can come
Derived from the various cartilaginous tissues such as articular cartilage, costal cartilage, Ear cartilage, nasal septal cartilage, cartilagines tracheales；Can also be with cartilage
The other cell types of human or animal of differentiation potential can derive from the tissues such as marrow, fat, muscle, skin.It is a kind of preferred next
Source is Ear cartilage, nasal cartilages costal cartilage from human or animal.
The method that separation obtains cartilage cell is the accepted method that document is repeatedly reported.A kind of preferred method be general anesthesia or
It is sterile under local anaesthesia to cut cartilaginous tissue, after PBS is cleaned, collagenase solution is added (concentration generally in 0.5-3mg/ml, with PBS
Or culture solution is prepared), 37 DEG C of constant temperature oscillations digest 4-20 hours (depending on the source of cartilaginous tissue and digestion progress extent), mistake
Cartilage cell's suspension is collected in filter, is counted under centrifugation, washing, Trypan Blue, microscope, and Primary chondrocyte vigor generally should be
80% or more.It is also the method for this field routine by the method that stem cell induces differentiation into cartilage cell.
Culture, propagating method and the culture solution of cartilage cell is also familiar in the field of competence.A kind of preferred method be by
Cartilage cell is in CO2Culture in incubator.Suitable culture solution includes (but being not limited to)：1) F-12 culture mediums or DMEM cultures
Base+5%-20% fetal calf serums；2) F-12 culture mediums or self (or allosome) human serums of DMEM culture mediums+5%-20%；3)F-
12/DMEM culture mediums (1:1)+2%-20% fetal calf serums or human serum.A kind of particularly preferred cartilage cell is in-vitro separation
The cartilage cell in 5 generation of 2nd generation-the of culture.Cartilage cell's function and vigor at this time is good, has very strong Subchondral drilling
Ability has II expression of collagen, RT-PCR and in situ hybridization detection to prove there are II Collagen Type VIs through immunocytochemical stain proof
And the expression of proteoglycans (aggrecan) mRNA.
Cartilage graft
Cartilage graft provided by the invention can be the cartilage diaphragm of external regeneration, can also be injectable cartilage transplanting
Object；Contain cartilage micro-assembly robot in the injectable cartilage graft.
Contain chondroblast in cartilage graft provided by the invention；The chondroblast is the 1st, 2,3,
4,5,6,7 or 8 generation；Preferably the 1st, 2,3,4 or 5 generation；More preferably it is 5 generation of 2nd generation-the.
Cartilage diaphragm provided by the invention is after chondroblast is carried out high density inoculation, by Fiber differentiation,
The sheet cartilaginous tissue of external regeneration, and cartilage micro-assembly robot provided by the invention is the cartilage diaphragm cutting that will be formed in this way and is obtained
It arrives.
In one embodiment of the invention, by the chondroblast Jing Guo above-mentioned amplification, passage with 3 × 105It is a
Cell/cm2-1×107A cell/cm2(preferably 1 × 106A cell/cm2-5×106A cell/cm2) density be inoculated in training
Ware is supported, chondrocytes in vitro Fiber differentiation is carried out, per changing the liquid once within 1-4 days, until regenerating sheet cartilaginous tissue in vitro.It will be regenerated
Cartilage diaphragm cuts or shreds to form cartilage micro-assembly robot.
Above-mentioned chondrocyte induction culture solution includes but not limited to, be added on the basis of regular growth culture solution TGF β 1, IGF and
Dexamethasone；More preferably, also contain and be selected from following one or more kinds of substances：Insulin, transferrins, bFGF, IBMX,
Sodium selenite, proline, seralbumin, beta -mercaptoethanol, L-Glutamine, linoleic acid and vitamin C phosphoric ester.
In the preferred embodiment of the present invention, the chondrocyte induction culture solution is on the basis of regular growth culture solution
1-50ng/ml TGF β 1,10-200ng/ml IGF and 10-200ng/ml dexamethasone is added；More preferably, it is additionally added following
One or more kinds of substances：1-50ng/ml insulin, 1-50mg/ml bFGF, 1-25ng/ml transferrins, 1-
20mmol/ml IBMX, 1-50ng/ml sodium selenites, 5 × 10-2- 1mg/ml proline, 1-100mg/ml seralbumins, 1-
20mg/ml beta -mercaptoethanols, 1-50mg/ml L-Glutamines, 1-20 μ g/ml linoleic acid and 10-200ng/ml vitamin C phosphorus
Acid esters.
The cartilage diaphragm of external regeneration provided by the invention can directly Hui Zhi in the defect of internal analogous shape, obtain
Engineered tissue or organ；Certain shape can also be formed in vitro, such as, but not limited to, tubulose, ear etc., then
Hui Zhi obtains engineered tissue or organ in the defect of internal analogous shape again.
In one embodiment of the invention, by the cartilage diaphragm of external regeneration using silicone tube as inner support, by two
Diaphragm is rolled into tubulose and is put into centrifuge tube, and the cartilage for obtaining external regeneration cartilage diaphragm that aforementioned present invention provides is added
After induction broth culture 1-10 weeks, silicone tube is removed, it is then that the obtained tubulose with certain mechanical strength and elasticity is soft
Bone returns corresponding defect in implant, obtains engineered tissue or organ.
Cartilage micro-assembly robot provided by the invention can obtain organizational project by Hui Zhi by way of injection in internal defect
Change tissue or organ.
Cartilage micro-assembly robot provided by the invention forms injectable cartilage graft after can also being mixed with injectable medium, so
By injectable cartilage graft, Hui Zhi obtains engineered tissue or device in internal defect by way of injection afterwards
Official；The weight ratio of the cartilage micro-assembly robot and injectable medium is 10:0-5.The injectable medium includes (but being not limited to)：
Physiological saline, PBS, autoserum, azelon gel, calcium alginate gel, hyaluronic acid, polyoxyethylene olefin(e) acid, dimethyl propylene
Alkene, polypropylene fumaric acid compound ethylene gel, polypropylene oxide etc.；It is preferred that physiological saline, hyaluronic acid or autoserum.
The present invention is using cartilage micro-assembly robot as syringeability cartilage graft.In one embodiment of the invention,
The cartilage micro-assembly robot can also mix that (weight ratio of cartilage micro-assembly robot and injectable medium is about with fraction of injectable medium
10:0-5), syringeability cartilage graft is formed.
Cartilage graft construction method
The preparation method of cartilage graft provided by the invention includes step：
Chondroblast is carried out plane culture amplification, and is passaged to by the first step, the acquisition and amplification of primary cell
1-8 generations；Preferably the 1st, 2,3,4 or 5 generation；More preferably it is 5 generation of 2nd generation-the；
Second step, the preparation of cartilage diaphragm, i.e., the cell inoculation expanded above-mentioned passage are placed in Nostoc commune Vanch ware
In chondrocyte induction culture solution cartilage diaphragm is formed through in vitro culture；
Third walks, and forms cartilage graft.
The method of plane culture amplification in the first step is such as, but not limited to, and isolated cartilage cell is resuspended in height
It in sugared DMEM culture solutions, then takes cell suspension inoculation on culture dish, cultivates under optimum conditions, wait for that cell growth is closely converged
Afterwards, it digests, (be such as, but not limited to, 1 in proportion:2-1:10；It is preferred that 1:3-1:7；More preferably it is 1:3-1:5) cell passage is carried out
To new culture dish, continue secondary culture under the same conditions.With the total volume meter of the DMEM in high glucose culture solution, preferably wherein
Contain 10-20%FBS, 1-10ng/ml bFGF；It is inoculated with about 0.5-2 × 10 in preferably every 10 cm dishes of inoculum concentration6A work
Cell.
Chondroblast is inoculated in after culture dish using chondrocyte induction culture solution in vitro culture until obtaining by second step
Cartilage diaphragm；For culture solution per changing the liquid once within 1-4 days, the external evoked time is 1-20 weeks, preferably 2-10 weeks.
In second step, chondroblast is with 3 × 105A cell/cm2-1×107A cell/cm2(preferably 1 × 106It is a thin
Born of the same parents/cm2-5×106A cell/cm2) density be inoculated in culture dish；The chondrocyte induction culture solution is in regular growth culture
1-50ng/ml TGF β 1,10-200ng/ml IGF and 10-200ng/ml dexamethasone are added on the basis of liquid；More preferably, may be used also
Following one or more kinds of substances are added：1-50ng/ml insulin, 1-50mg/ml bFGF, 1-25ng/ml turn iron egg
In vain, 1-20mmol/ml IBMX, 1-50ng/ml sodium selenites, 5 × 10-2- 1mg/ml proline, the white egg of 1-100mg/ml serum
In vain, 1-20mg/ml beta -mercaptoethanols, 1-50mg/ml L-Glutamines, 1-20 μ g/ml linoleic acid and 10-200ng/ml dimension lifes
Plain C phosphates.
In one embodiment of the invention, the cartilage diaphragm for making second step obtain in above-mentioned third step forms different
The cartilage graft of shape；Such as, but not limited to, tubulose cartilage graft is formed by inner support of cylindrical silicone tube.
In another embodiment of the invention, the cartilage diaphragm that second step obtains is cut or cut in above-mentioned third step
It is broken, form cartilage micro-assembly robot；The cartilage diaphragm that second step obtains is cut into 0.5-1.5mm*0.5- in one embodiment
After 1.5mm sizes, the physiological saline or PBS of 1-10 times of volume (preferably 3-5 times of volume) is added, centrifugation is absorbed supernatant, obtained
Cartilage micro-assembly robot, you can injection cartilage graft.
Can the cartilage diaphragm after cleaning be subjected to Hui Zhi, the cartilage micro-assembly robot after cleaning can also be mixed with injectable medium
Form injectable cartilage graft.The injectable medium includes but is not limited to physiological saline, PBS, autoserum, albumen
Fiber gel, calcium alginate gel, hyaluronic acid, polyoxyethylene olefin(e) acid, dimethyl allene, polypropylene fumaric acid compound ethylene are solidifying
Glue, polypropylene oxide etc.；It is preferred that physiological saline, hyaluronic acid or autoserum；The weight of cartilage micro-assembly robot and injectable medium
Than being 10:0-5.
Above-mentioned cleaning operation can remove the various growth factors being adsorbed in incubation on diaphragm, to eliminate by these
Growth factor and the organism immune response (such as swelling) caused.Cleaning may be repeated, such as, but not limited to, 1-5 times, and preferably
2-4 times.
The cartilage diaphragm or syringeability cartilage graft of the specific shape of formation are injected into vivo, to repair damaged part
Or filling body surface recess.
Using
Injectable cartilage graft provided by the invention can be adapted for the reparation of various cartilage defects, including (but not
It is limited to) such as：Joint, tracheae, ear, all kinds of cartilage defect repairs such as nose；And minimally invasive filling beauty and shaping, including (but and it is unlimited
In) such as：Augmentation rhinoplasty, grand lower chin, rich temples etc..
The feature that the features described above or embodiment that the present invention mentions are mentioned can be in any combination.Disclosed in this case specification
All features can be used in combination with any composition form, each feature disclosed in specification, any can provide it is identical,
The alternative characteristics of impartial or similar purpose replace.Therefore it is only impartial or similar spy except having special instruction, revealed feature
The general example of sign.
Main advantages of the present invention are：
(1) a small amount of self cartilage of application realizes the regeneration of large volume cartilage：It (is less than using a small amount of self cartilaginous tissue
100mg) obtain cartilage cell and large amplification；It is special at chondrocyte induction system to be combined by high density inoculation, reverses high generation
Secondary cartilage cell's dedifferen tiation, external regeneration forms large volume cartilage (being more than 10g), small for area's wound.
(2) chondrocytes in vitro regenerates, safely controllable：1. simple cartilage cell's external regeneration cartilaginous tissue, no timbering material ginseng
With and interference, regenerating bone or cartilage is technically simple controllable, and stability is good.2. can autogenous cell source, during external regeneration cartilage not
It is safe containing the animals derived components such as serum.3. regenerating bone or cartilage process is mainly completed in vitro, with standardized production and can carry out
Assessment and detection before the implantation of system, such as：Histological stain, collagen, aseptic detection etc..Production process and regeneration of cartilage
Quality safety is controllable.
(3) internal regenerating bone or cartilage effect stability：1. the technology preliminary in vitro, which builds the cartilage diaphragm to be formed, contains a large amount of spies
Sign property extracellular matrix, has been more mature cartilaginous tissue；2. the technology is participated in without timbering material, avoid caused by material not
Good inflammatory reaction influences internal regenerating bone or cartilage.3. in big animal model body when the cartilage graft of autologous transplanting cytothesis,
The cartilage volume formed for postoperative 1 year is transplant initial volume 80% or more, effect stability and result with controllability.Cartilage
Forming insufficient region can completely be repaired by way of secondary supplement transplanting.
(4) easily moulding and holding can be stablized：Solid cartilage diaphragm or cartilage micro-assembly robot mobility are poor, be easy it is moulding and
Holding can be stablized.
(5) incidence of complications is low：Cartilage diaphragm or the transplanting of cartilage micro-assembly robot will not enter blood vessel, and complication occurs
Rate is extremely low.
(6) clinical effectiveness is lasting：Self cartilage and long-term surviving can be formed, clinical effectiveness can be maintained permanently.
(7) advantage is notable compared with similar technique：Chondrocytes in vitro regeneration techniques provided by the invention are than prior art step
Simplify, effect is more stablized.1. (such as with syringeability filler：Hyaluronic acid, synthetic or animal derived collagen etc.) phase
Than curative effect is more longlasting；2. with cartilage cell compared with degradation material compound, curative effect is stably and controllable, safe；3. with existing
Some diaphragm technologies of preparing are compared, easier, stable, safety.
Below in conjunction with specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate this hair
It is bright rather than limit the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to routine
Condition, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no
Then all percentages and parts are counted by weight.
The unit in percent weight in volume in the present invention is well-known to those skilled in the art, for example, refer to
The weight of solute in 100 milliliters of solution.
Unless otherwise defined, all professional and scientific terms used in text and meaning known to one skilled in the art
Justice is identical.In addition, any method and material similar or impartial to described content can be applied to the method for the present invention.Wen Zhong
The preferred implement methods and materials are for illustrative purposes only.
5 goats, asexuality requirement, average weight 20.0kg ± 3kg are used in following embodiments.It is protected according to experimental animal
Finger shield south, all animals all obtain humanitarian treat.
Embodiment 1
The acquisition and amplification of primary cell
Every animal takes the auricular cartilage of about 1.0cm x1.0cm sizes, and cartilage block is shredded, and 0.25% tryptose is added
Enzyme (Hyclone companies of the U.S.), concussion digestion 30min, is then rinsed 3 times with PBS, the 0.25% II Collagenase Type (U.S. is added
Worthington companies), concussion digestion 7-8h.100 micrometer cell strainer filterings of the digestive juice of acquisition, centrifugation, remove supernatant
The cell of liquid, precipitation is resuspended with high sugared Dahl Burke Improved Eagle Medium (DMEM, Gibco companies of the U.S.), gained cell
With 1x104The concentration of/cm2 is seeded on 100mm culture dishes, at 37 DEG C, 5%CO2, under the conditions of saturated humidity, with containing 10% tire ox
DMEM culture medium (the cultured chondrocytes of serum (Hyclone companies of the U.S.), 2ng/mlbFGF, 1% penicillin and streptomysin
Liquid) culture amplification, it is passed on when chondrocyte growth to 70%-80% merges.
Embodiment 2
Prepare cartilage diaphragm 1
The P4 that embodiment 1 is obtained is digested for cartilage cell, is centrifuged, and is resuspended, with 1x106/cm2Concentration is inoculated in common training
It supports on ware, with chondrocyte induction culture solution (1-50ng/ml insulin, 1-50mg/ml bFGF, 1-25ng/ml transferrins, 1-
20mmol/ml IBMX, 1-50ng/ml sodium selenites, 5 × 10-2- 1mg/ml proline, 1-100mg/ml seralbumins, 1-
20mg/ml beta -mercaptoethanols, 1-50mg/ml L-Glutamines, 1-20 μ g/ml linoleic acid and 10-200ng/ml vitamin C phosphorus
Acid esters, 10ng/ml dexamethasone, 5ng/m TGF β 1 and 5ng/ml IGF) culture, liquid is changed daily.It can be with when in vitro culture 4 weeks
The cartilage diaphragm 1 that a 100mm diameters are uncovered in culture dish carries out assessment and detection before the implantation of system, such as：Volume weight in wet base is surveyed
Amount, histological stain, collagen, aseptic detection etc..As a result, it has been found that cartilage membrane body product weight in wet base is smaller, explanation forms
Relatively thin cartilage diaphragm tissue.
Embodiment 3
Prepare cartilage diaphragm 2
The P4 that embodiment 1 is obtained is digested for cartilage cell, is centrifuged, and is resuspended, with 2x106/cm2Concentration is inoculated in common training
It supports on ware, with chondrocyte induction culture solution (1-50ng/ml insulin, 1-50mg/ml bFGF, 1-25ng/ml transferrins, 1-
20mmol/ml IBMX, 1-50ng/ml sodium selenites, 5 × 10-2- 1mg/ml proline, 1-100mg/ml seralbumins, 1-
20mg/ml beta -mercaptoethanols, 1-50mg/ml L-Glutamines, 1-20 μ g/ml linoleic acid and 10-200ng/ml vitamin C phosphorus
Acid esters, 10ng/ml dexamethasone, 5ng/m TGF β 1 and 5ng/ml IGF) culture, liquid is changed daily.It can be with when in vitro culture 4 weeks
The cartilage diaphragm 2 that a 100mm diameters are uncovered in culture dish carries out assessment and detection before the implantation of system, such as：Volume weight in wet base is surveyed
Amount, histological stain, collagen, aseptic detection etc..As a result, it has been found that cartilage membrane body product weight in wet base is bigger than normal, explanation forms
The larger cartilage diaphragm tissue of thickness.
Embodiment 4
Using containing only TGF β 1, the chondrocyte induction culture solution in vitro culture of IGF and dexamethasone prepares cartilage diaphragm
The P4 that embodiment 1 is obtained is digested for cartilage cell, is centrifuged, and is resuspended, with 5x106/cm2Concentration is inoculated in common training
It supports on ware, is cultivated with chondrocyte induction culture solution (1-50ng/ml TGF β 1,5ng/ml IGF and 10ng/ml dexamethasone), daily
Change liquid.The cartilage diaphragm that a 100mm diameters can be uncovered when in vitro culture 4 weeks in culture dish, is commented before carrying out the implantation of system
Estimate and detect, such as：Volume weight in wet base measures, histological stain, GAG and collagen, aseptic detection etc..As a result, it has been found that application should
The cartilage diaphragm that induction broth in vitro culture is formed has met implantation and has required, although volume, weight in wet base, GAG and collagen content are inclined
It is small.
Embodiment 5
Tubulose cartilage is built outside cartilage membrane body
Two cartilage diaphragms superposition that embodiment 2 is obtained, at 37 DEG C, 5%CO2, 30 points are placed under the conditions of saturated humidity
Two diaphragms are rolled into tubulose and are put into 50ml centrifuge tubes by Zhong Hou using silicone tube as inner support, and 40ml chondrocyte inductions are added
Culture solution (1-50ng/ml insulin, 1-50mg/ml bFGF, 1-25ng/ml transferrins, 1-20mmol/ml IBMX, 1-
50ng/ml sodium selenites, 5 × 10-2- 1mg/ml proline, 1-100mg/ml seralbumins, 1-20mg/ml β-sulfydryl second
Alcohol, 1-50mg/ml L-Glutamines, 1-20 μ g/ml linoleic acid and 10-200ng/ml vitamin C phosphoric esters, fill in 10ng/ml
Meter Song, 5ng/m TGF β 1 and 5ng/ml IGF) culture, liquid is changed daily.In vitro culture can obtain having certain mechanics after 8 weeks
The tubulose cartilage of intensity and elasticity.Referring to the G in attached drawing 1.
Embodiment 6
Sheet cartilage is formed in cartilage membrane body, structure in-vivo tissue is engineered tracheae
The self cartilage diaphragm that embodiment 2 obtains is implanted to sheep subcutaneous abdomen.The postoperative 8 weeks pieces for obtaining about 90mm diameters
Shape cartilage can build to form functional organization's engineering tracheae in vivo after Prevascularized and epithelialization.Referring in attached drawing 1
H.
Embodiment 7
The preparation and cleaning of injectable cartilage micro-assembly robot
The cartilage diaphragm that embodiment 2 obtains is cut into 1.0mm × 1.0mm sizes, be added 3 times of volumes physiological saline or
PBS is centrifuged with 1000 revs/min, absorbs supernatant for * 5 minutes.Repeat the above cleaning operation 3 times.
Embodiment 8
The internal injection of simple cartilage micro-assembly robot
Cartilage micro-assembly robot after the cleaning that embodiment 3 obtains is injected into abdomen skin as syringeability cartilage graft
Under.
Embodiment 9
The internal injection of simple cartilage micro-assembly robot
Cartilage micro-assembly robot after the cleaning that embodiment 3 is obtained and a small amount of hyaluronic acid or physiological saline (about 10:1 ratio
Example) it is mixed to form syringeability cartilage graft and is injected into subcutaneous abdomen.24w materials observations.The result shows that substantially seeing can be shown in Table
Face porcelain white, the stronger chondroid tissue of hardness.Histology HE dyeing is further confirmed that it is the cartilage sample of homogeneous maturation
Tissue, specific cartilage cavities spline structure are uniformly distributed, and contain a large amount of extracellular matrixs.Referring to the E in attached drawing 1, F.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (10)
1. a kind of cartilage graft, which is characterized in that the cartilage graft contain external regeneration cartilage diaphragm or cartilage it is micro-
Tissue.
2. cartilage graft as described in claim 1, which is characterized in that the cartilage graft contains cartilage micro-assembly robot and can
Injectable media；The weight ratio of the cartilage micro-assembly robot and injectable medium is 10:0-5；The injectable medium is selected from physiology salt
Water, phosphate buffer (PBS), hyaluronic acid or autoserum.
3. cartilage graft as claimed in claim 1 or 2, which is characterized in that the external regeneration cartilage diaphragm or cartilage are micro-
Tissue is prepared by following step：
(1) it cartilage cell's cultured and amplified in vitro and passes on；
(2) chondrocyte induction culture is carried out after being inoculated with the cartilage cell of passage, obtains the cartilage diaphragm of external regeneration；
(3) regenerated cartilage diaphragm obtains cartilage micro-assembly robot outside cutting body.
4. cartilage graft as claimed in claim 3, which is characterized in that cartilage cell's inoculum density in step (2) is 3 ×
105A cell/cm2-1×107A cell/cm2；It is preferred that 1 × 106A cell/cm2-5×106A cell/cm2。
5. cartilage graft as claimed in claim 3, which is characterized in that contain 1- in the culture solution of the chondrocyte induction culture
50ng/ml transforming growth factor (Transforming growth factor beta 1, TGF β 1), 10-200ng/ml pancreas islet
Plain like growth factor (Insulin like growth factor, IGF) and 10-200ng/ml dexamethasone.
6. cartilage graft as claimed in claim 3, which is characterized in that the cartilage cell be the 1st, 2,3,4,5,6,7 or
8 generation secondary cultures；It is preferred that 5 generation of 2nd generation-the.
7. cartilage graft as claimed in claim 3, which is characterized in that the cartilage cell is from self or of the same race different
Body；It is preferred that Auricular cartilage, nasal septum and costal cartilage.
8. a kind of preparation method of such as claim 1-7 any one of them cartilage grafts, which is characterized in that the method
Including step：
(1) cartilage cell is subjected to plane amplification, and is passaged to the generation of the 1st, 2,3,4,5,6,7 or 8；
(2) cartilage cell's high density that the generation of the 1st, 2,3,4,5,6,7 or 8 passes on is seeded on Tissue Culture Dish and carries out cartilage
Fiber differentiation obtains external regeneration cartilage diaphragm；
(3) the outer regeneration of cartilage diaphragm of cutting body forms cartilage micro-assembly robot；
(4) claim 1-7 such as is obtained after cleaning external regeneration cartilage diaphragm or cartilage micro-assembly robot physiological saline or PBS to appoint
Cartilage graft described in one.
9. a kind of such as claim 1-7 any one of them cartilage graft answering in building organization engineered cartilage tissue
With.
10. a kind of cartilage cell builds the application in cartilage graft in vitro.
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| CN109893301A (en) * | 2019-04-23 | 2019-06-18 | 上海市第六人民医院 | A kind of transplant and method for repairing articular cartilage defect |
| WO2022156648A1 (en) * | 2021-01-20 | 2022-07-28 | 上海软馨生物科技有限公司 | Ear cartilage tissue engineering complex and use thereof |
| WO2022156645A1 (en) * | 2021-01-20 | 2022-07-28 | 上海软馨生物科技有限公司 | Cartilage tissue engineering complex and use thereof |
| WO2022156769A1 (en) * | 2021-01-22 | 2022-07-28 | 上海软馨生物科技有限公司 | Large aperture-based tissue engineering scaffold and use thereof |
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| CN104922730A (en) * | 2015-04-29 | 2015-09-23 | 陕西瑞盛生物科技有限公司 | Cartilage cell tissue engineering material and preparation method and application thereof |
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| CN1306085A (en) * | 1999-08-19 | 2001-08-01 | 泽恩比奥公司 | Application of substrate cell derived from fat tissue |
| CN104922730A (en) * | 2015-04-29 | 2015-09-23 | 陕西瑞盛生物科技有限公司 | Cartilage cell tissue engineering material and preparation method and application thereof |
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| CN109893301A (en) * | 2019-04-23 | 2019-06-18 | 上海市第六人民医院 | A kind of transplant and method for repairing articular cartilage defect |
| WO2022156648A1 (en) * | 2021-01-20 | 2022-07-28 | 上海软馨生物科技有限公司 | Ear cartilage tissue engineering complex and use thereof |
| WO2022156645A1 (en) * | 2021-01-20 | 2022-07-28 | 上海软馨生物科技有限公司 | Cartilage tissue engineering complex and use thereof |
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Effective date of registration: 20190820 Address after: No. 68 Jiangchuan East Road, Minhang District, Shanghai, 200241 Applicant after: Shanghai Soft Heart Biotechnology Co., Ltd. Address before: No. 68 Jiangchuan East Road, Minhang District, Shanghai, 200241 Applicant before: Cao Yilin |