CN107320778A - A kind of preparation method of cartilage acellular matrix - Google Patents
A kind of preparation method of cartilage acellular matrix Download PDFInfo
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- CN107320778A CN107320778A CN201710724873.0A CN201710724873A CN107320778A CN 107320778 A CN107320778 A CN 107320778A CN 201710724873 A CN201710724873 A CN 201710724873A CN 107320778 A CN107320778 A CN 107320778A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
- A61L27/3645—Connective tissue
- A61L27/3654—Cartilage, e.g. meniscus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Abstract
The present invention relates to Autologous Chondrocyte implantation technique field, more particularly to a kind of preparation method of cartilage acellular matrix.The preparation method that the present invention is provided carries out acellular presence in cell removing, the cartilage acellular matrix obtained, DNA content is extremely low, and the content of collagen is then unaffected using organization engineered cartilage as raw material.The integrality of good cartilaginous tissue is maintained, biological structure is good, and better performance can be had by being used as support.
Description
Technical field
The present invention relates to Autologous Chondrocyte implantation technique field, more particularly to a kind of preparation side of cartilage acellular matrix
Method.
Background technology
Normal articular cartilage be hyalomitome cartilage, be it is a kind of have very strong tolerance and the single tissue of structure.By various
Osteoarticular injury or cartilage defect caused by the different types of cause of disease are orthopedics and traumatology common disease, are mainly shown as arthralgia, swell
Swollen, stiff and dysfunction, and can gradually occur retrogression.Wherein knee articular cartilage defect is common in osteoarticular injury
Disease.Knee articular cartilage defect is worldwide fairly common, there is foreign scholar's statistics, is doing the trouble of arthroscopy of knee
In person, there is more than 60% to there is articular cartilage damage.Due to no nerve and vascular tissue, the nutrition of cartilage is relied primarily on from cunning
Absorbed in the synovia of film secretion.But because proteoglycan synthesis is suppressed, collagenous fibres are destroyed, and make its bullet of cartilage loss
Articular cartilage damage mechanism, the compression for adding hydraulic permeation and bearing cartilage cell increases, and causes catabolic enzyme to increase
Plus, lubricious effect declines and causes articular cartilage surface to destroy.Articular chondrocytes after damage are because its metabolism is slow, it is difficult to certainly
Row is repaired, even if smaller cartilage damage may also produce more significant symptom, ultimately results in the degenerative change in joint.
The reparation of articular cartilage is by patient age, damaged part, defect scope, depth, the stability in joint, injured journey
Many-sided influence such as degree.Autologous Chondrocyte transplanting is suitable for relating only to caused by acute or chronic wound cartilage surface and cartilage
The damage of sending down the fishbone completely.This method is for small areas cartilage damage patient is in pain of alleviation and improves function aspects with preferable
Curative effect.Compared with other method such as micro fractures method, Autologous Chondrocyte transplantation can produce more hyaline cartilage sample reparation
Tissue, and with more preferable clinical effectiveness, the technology has become the most important operation method for the treatment of knee cartilage lesion
One of.But this operation is related to coating and viscous seam, adds the complexity of operation；The transplanting of third generation Autologous Chondrocyte has branch
Frame material, the problem of biocompatibility occurs in exogenous material；Postoperative complication occurs；Simultaneously because Autologous Chondrocyte meeting
It is adherent, cause part cartilage cell to dedifferente to generate fibrocartilage, but still fail preferably to cartilage defect area to hold
Continue stable mode row hyaline cartilage to repair, so as to can not preferably recover the good biomechanical property of articular cartilage.
Also, why autologous hyaline cartilage (LhCG), which can treat injury of knee joint, is mainly autologous hyaline cartilage filling
Defects of knee, when being transplanted into animal body, the only proteoglycan calcium and II Collagen Type VIs of living cells and its secretion.
Mechanical strength is a relatively soft, spongiform state between current product and natural cartilage, can be soft with surrounding
When 2 months or so in the good syntrophism of bone tissue, transplanting animal body, it is possible to hard as real cartilage.But into
Ripe Autologous Chondrocyte amplification is difficult, and autologous cartilaginous tissue limited source, materials are inconvenient, and in vitro culture is easily dedifferented
Phenomenon, loses Subchondral drilling ability.And its activity increases and is decreased obviously with the age so that cell amplification is restricted, up to not
To the requirement of tissue construction.It is difficult to be generalized to clinic that these problems, which limit the autologous hyaline cartilage of tissue engineering product (LhCG),
On.
At present, the method for more promising labor articular cartilage disease is exactly that largely injection mesenchymas are dry thin in articular cavity
Born of the same parents, because cell can start regeneration activity as the bioactie agent of nutrition causes the reparation of defect.Cultured chondrocytes
Fixation problem after condition and transplanting limits the clinical practice of chondrocyte cell transplantation.Have research first by cartilage cell plant in
On artificial synthesized polymer and collagen gel support, cartilaginous tissue has successfully been regenerated in vitro, has been lacked for repairing articular cartilage
Damage, obtain satisfied effect.But after cytoskeleton compound is implanted into vivo, support will likely cause receptor's immune
Rejection；Original tissue and timbering material integrate difficulty, and the mechanical strength of compound is relatively low.
Acellular matrix (acellular matrix, ACM) refers generally to after allosome tissue handles through cell inactivation, be prepared into
The ECM compositions and structure existed without active somatic cell.Because of its no antigen, and there is good biocompatibility, as tissue work
One of selection of engineering support material.The corium of de- cell processing, pericardium, cornea, blood vessel, bladder, oesophagus, mucous membrane of small intestine matrix,
Bone, liver etc. have been succeeded in developing in succession as the substitute of respective organization, show good application prospect.By in cartilaginous tissue
Cell remove as cytoskeleton, the problem of rejection being solved, as the tissue work with good biocompatibility
Engineering support material.Yet with autologous cartilaginous tissue limited source, and the removing difficulty of cell is larger, so autologous cartilage is removed
The timbering material that cell is made is very rare.
The content of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of preparation method of cartilage acellular matrix,
The method that the present invention is provided carries out de- cell to tissue engineering bone/cartilage, and the de- cellular cartilage obtained is taken off after cell with autologous cartilage
Performance it is consistent.
The preparation method for the cartilage acellular matrix that the present invention is provided, for by tissue engineering bone/cartilage successively through A liquid, B liquid at
After reason, digested, then handled through C liquid with RnaseA and Dnase I, obtain de- cellular cartilage；
The A liquid is the mass fraction of Tris-HCl buffer solutions, wherein protease inhibitors containing protease inhibitors
For 0.01%~1%；
The B liquid is the quality of Tris-HCl buffer solutions, wherein protease inhibitors containing SDS and protease inhibitors
Fraction is 0.01%~1%；SDS volume fraction is 2%；
The C liquid is the volume fraction of Tris-HCl buffer solutions, wherein Triton X-100 containing Triton X-100
For 1%.
In the Tris-HCl buffer solutions of the A liquid, Tris-HCl concentration is 1~100mM；PH value is 5.0~8.
In the Tris-HCl buffer solutions of the B liquid, Tris-HCl concentration is 1~100mM；PH value is 5.0~8.
In the Tris-HCl buffer solutions of the C liquid, Tris-HCl concentration is 1~100mM；PH value is 5.0~8.
In some embodiments, the condition of A liquid processing is 0~4 DEG C, concussion processing 48h.
In some embodiments, the condition of B liquid processing is 0~4 DEG C, concussion processing 48h, then with distilled water flushing 24h.
In some embodiments, in RnaseA and Dnase I enzymolysis enzymolysis, RnaseA amount is 1g/L；Dnase I amount is
500U/mL。
In some embodiments, the condition of C liquid processing is 0~4 DEG C, 24h is digested, then with distilled water flushing 24h.
Due to natural tissues (autologous cartilage) compact structure, thus for autologous cartilage cell free method be will be autologous
Cartilage is broken into the posterior synechia fragment of fritter, destroys the integrality and internal structure of tissue；And use tissue engineering bone/cartilage to take off
Cell can thoroughly be carried out on the premise of graft integrality is not destroyed.
Also, because its biological structure has been destroyed, and it can not recover, autologous cartilage is taken off after cell, need to be broken through that will organize
Block carries out adhesion, with the graft of formation；But the graft of this Adhesion formation does not have compared to synthetic material support
Clear superiority.And the de- cell of tissue engineering bone/cartilage can keep the original biological structure of tissue engineering bone/cartilage, it is not required to carry out adhesion,
Performance is also superior to synthetic material.
De- cell is carried out using tissue engineering bone/cartilage to handle, the problem of autologous cartilage limited source can be solved, and due to
Remove after cell, it is to avoid the generation of immune response, can clinically realize allogeneic even heterograft.Allosome/xenogenesis
Transplanting can reduce the operation number of times of patient.General knee joint prosthesis need to perform the operation twice at present, and first time surgical collection is thin
Born of the same parents, second of operation implantation autograft thing.Operation can be reduced to once by allosome/heterograft.It can avoid supplying simultaneously
The problem of body region necrosis or secondary infection.
But the tissue engineering bone/cartilage of cellar culture and autologous cartilage are distinct, effect is used as support after removing cell
Fruit is not good, so the preparation method of the tissue engineering bone/cartilage of the application offer is provided.
The tissue engineering bone/cartilage that the present invention is used is pig source tissue engineered cartilage, and it is that pig source cartilaginous tissue is obtained through culture
.
It is preferred that, the preparation method of tissue engineering bone/cartilage is：
Step 1：After autologous cartilage is digested with II Collagen Type VIs, original cuiture is carried out with CM culture mediums；
Step 2：After cell after original cuiture is with Trypsin Induced, it is resuspended with sodium alginate soln；
Step 3：The cell of resuspension is connected to the coated culture vessel of gelatin, 0~4 DEG C is placed after 4min, adds CaCl2It is molten
Liquid, 0~4 DEG C of placement 4min, forms Cellular gels；
Step 4：Cellular gels are transferred to the coated culture vessel of agarose, with CC medium cultures 15~25 days, training
Change fresh culture within every 2~3 days during supporting；Obtain tissue engineering bone/cartilage.
The primary culture method of autologous cartilage is：By cartilaginous tissue it is sterilized after be placed in CM culture mediums, after chopping, reject
CM culture mediums, with 1mg/mLII Collagenase Type solution, 37 DEG C, 50rpm digests 14h；Then the digestive juice of cartilaginous tissue will be digested
1100rpm centrifuges 5min, removes supernatant；Precipitation is resuspended with CM culture mediums, 37 DEG C, 5%CO2, saturated humidity culture 8 days, culture
During change fresh CM culture mediums daily.Change before fresh culture with PBS cleaning cell.Culture is soft to the 8th day
Osteocyte degrees of fusion reaches 95%, passage.
It is described passage refer to by the cell of original cuiture with Trypsin Induced after, switching continue cultivate process.
Trypsin Induced is specially：Mass fraction is 0.25% trypsase, 37 DEG C, digests 4min.
After digestion, digested with CM culture mediums germplasm, 1100rpm, 5min centrifuge cells remove supernatant.It is precipitated as cartilage cell.
Cartilage cell is resuspended with sodium alginate soln.Culture vessel preferably 24 orifice plates.Using concentration as 1%~50% it is bright
Coated 24 orifice plate of glue；The orifice plate of agar glycolyx 24 using concentration as 0.1%~12%.
In the embodiment of the present invention, the mass fraction of the middle sodium alginate of sodium alginate soln is 0.1%~20%.
Preferably, the mass fraction of the middle sodium alginate of sodium alginate soln is 1.5%.
In the embodiment of the present invention, it is 2 × 10 to be resuspended to the density of the middle cell of sodium alginate soln6Individual cell/mL.
Sodium alginate is resuspended after cell, is added in coated 24 orifice plate of gelatin, per the μ L cell suspensions of hole 400, toward culture plate
Added in the middle of hole, it is to avoid produce bubble, rotating and culturing plate so that cell suspension is paved with hole.
In the embodiment of the present invention, CaCl2The concentration of solution is 200mmol/L；CaCl2The body of solution and sodium alginate soln
Product is than being 1：1.
CaCl2Solution feed postition rotatably adds to be light and slow on hole wall.
Add 1mLCC culture mediums per hole into 24 orifice plates of coating agarose in advance.The cell of solidification is coagulated with small spoon
Blob of viscose is transferred in coated 24 orifice plate of agarose and cultivated.Liquid was changed every 2~3 days, co-cultures 20 days, can obtain transparent soft
Bone.
The method provided using the present invention, cartilage cell is wrapped in dimensional culture in Sodium Alginate Hydrogel Films, culture to the 3rd
My god, cell growth is not obvious, such as Fig. 2.But when cell culture was to the 20th day, it can be seen that base is constantly bred and secreted to cell
Matter, in histioid form, such as Fig. 3.
Experiment shows that the matrix prepared using the method provided by the present invention is not taken off compared with not cell free hyalomitome cartilage
Cytohyalop lasm cartilage is consistent with the II Collagen Type VI contents of de- cytohyalop lasm cartilage, but in de- groups of cells inside cartilage cavities
The presence of nucleus is can't see, and de- groups of cells can significantly see the presence of nucleus, that is, the hyalomitome for taking off groups of cells is soft
Osteocyte is taken off.Separately quantified from DNA and toluene blue dyeing liquor can be seen that, it is very complete that cell is removed.
Cartilage acellular matrix made from preparation method of the present invention.
The preparation method that the present invention is provided carries out cell removing, the cartilage obtained using organization engineered cartilage as raw material
Acellular in acellular matrix to exist, DNA content is extremely low, and the content of collagen is then unaffected.Maintain good soft
The integrality of bone tissue, biological structure is good, and better performance can be had by being used as support.
Brief description of the drawings
Fig. 1 show pig source cartilage cell's original cuiture to the 8th day 100 ×；
Fig. 2 show the pig source cartilage cell's dimensional culture of embodiment 2 to the 3rd day 100 ×；
Fig. 3 show the pig source cartilage cell's dimensional culture of embodiment 2 to the 20th day 100 ×；
Fig. 4 show organizational project hyalomitome cartilage II Collagen Type VIs dyeing 200 ×；
Fig. 5 show organizational project hyalomitome cartilage Toluidine blue staining 200 ×；
Fig. 6 show organizational project hyalomitome cartilage HE dyeing 200 ×；
Fig. 7 show organizational project hyalomitome cartilage acellular matrix II Collagen Type VIs dyeing 200 ×；
Fig. 8 show organizational project hyalomitome cartilage acellular matrix Toluidine blue staining 200 ×；
Fig. 9 show organizational project hyalomitome cartilage acellular matrix HE dyeing 200 ×；
Figure 10 shows that tissue engineering bone/cartilage, organizational project acellular matrix, the DNA content of natural cartilage acellular matrix are surveyed
It is fixed；
Figure 11 show the natural cartilage acellular matrix II Collagen Type VIs of comparative example 1 dyeing 200 ×；
Figure 12 show the natural cartilage acellular matrix Toluidine blue staining 200 of comparative example 1 ×；
Figure 13 show the natural cartilage acellular matrix HE of comparative example 1 dyeing 200 ×；
Figure 14 show the conventional organization engineered cartilage type i collagen of comparative example 2 dyeing 200 ×；
Figure 15 show the conventional organization engineered cartilage II Collagen Type VIs of comparative example 2 dyeing 200 ×.
Embodiment
The invention provides a kind of preparation method of cartilage acellular matrix, those skilled in the art can be used for reference in herein
Hold, be suitably modified technological parameter realization.In particular, all similar replacements and change are to those skilled in the art
For be it will be apparent that they are considered as being included in the present invention.The method of the present invention and application are by preferably implementing
Example is described, and related personnel substantially can be not departing from present invention, in spirit and scope to methods herein and application
It is modified or suitably change is with combining, realizes and apply the technology of the present invention.
The examination material that the present invention is used is all common commercially available product, can all be bought in market.
With reference to embodiment, the present invention is expanded on further：
The cartilage original cuiture of embodiment 1
Fresh pig cartilage is divided into multiple fritters with scalpel, prizes by one piece of block cartilage from the bottom up, it is placed in 1 × it is bis-
In anti-PBS solution.It is quickly transferred to after weighing in super-clean bench.
1 × dual anti-PBS is substituted for 10 × dual anti-PBS solution, firmly rocks the centrifuge tube equipped with cartilage, continue
10s.Cartilage is transferred in 100mm culture dish.Discard solution with 1ml pipette tips, 1 new × dual anti-PBS of addition,
The inner surface of cartilage piece is scraped totally with clean a scalpel and tweezers, PBS is discarded.
10ml CM culture mediums are added in clean cartilage piece to handling, cartilage piece is cut into the fragment of grain of rice size, is placed in
Wait to digest in incubator.CM culture mediums are discarded, 15mL 1mg/mLII Collagenase Type solution is added.It is placed in incubator on shaking table,
37 DEG C of digestion 14h (50rpm).
Digestion terminates, and the digestive juice for digesting cartilaginous tissue is transferred in 50mL centrifuge tubes with 1mL pipette tips.Cell count
Afterwards, 1100rpm centrifuges 5min, removes supernatant, adds CM culture mediums.It is seeded in culture dish, is placed in incubator and cultivates.Inoculation
The 2nd day, discard cultured chondrocytes base, with PBS rinse one time, addition culture medium continue cultivate.Cultivate to the 8th day, cartilage
Cell fusion degree reaches 95%, as shown in Figure 1.
The preparation and detection of the tissue engineering bone/cartilage of embodiment 2
First, cartilage cell's dimensional culture
(1) agar glycolyx culture plate
Agarose spreads 24 orifice plates with preceding heating for dissolving, is placed in 4 DEG C of preservations, is used after solidification use.
(2) gelatin substrate coating culture dish
Gelatin substrate is spread 24 orifice plates, is placed in 4 DEG C of preservations with preceding heating for dissolving.Used after solidification.
(3) cartilage cell is digested
Trypsase is placed in 37 DEG C of preheatings in advance, digests cartilage cell, 0.25% Trypsin Induced 4min.Cultivated with CM
Base and trypsase 1：1 ratio terminates digestion.Cell is counted with tally, 1100rpm, 5min centrifuge cells, removes supernatant.
(4) cell is resuspended with sodium alginate soln
With 2 × 106The ratio of individual cell/mL sodium alginate solns (mass fraction is 1.5%, and viscosity is 20~500cps)
Example, sodium alginate soln is added into cell, and cell is resuspended.
(5) cell suspension is added in coated 24 orifice plate of gelatin substrate, per the μ L cell suspensions of hole 400, toward culture plate
Added in the middle of hole, it is to avoid produce bubble, rotating and culturing plate so that cell suspension is paved with hole.It is placed in 4 DEG C, 4min.
(6) take out culture plate after 4min, to the hole wall for have cell on light and slow rotatably add 400 μ L 200mM CaCl2It is molten
Liquid/per hole.It is placed in 4 DEG C, 4min.
(7) 1mL CC culture mediums are added per hole into 24 orifice plates of coating agarose in advance.With small spoon by the thin of solidification
Born of the same parents' gel piece is transferred in coated 24 orifice plate of agarose and cultivated.Liquid was changed every 2~3 days, co-cultures 20 days, can obtain transparent
Cartilage.As shown in Figures 2 and 3.
2nd, the identification of hyaline cartilage
1. II Collagen Type VIs SABC detects the content and the situation of II Collagen Type VIs of nucleus in cartilage：
1.4 μm of paraffin sections routinely dewax to water；2.PBS washes 3 × 3min；3.3%H2O2 room temperatures 20min, PBS wash 3 ×
3min；4. 4% stomach cardia alcohol 60min, PBS of digestion washes 3 × 3min；5. primary antibody 1:50,4 DEG C overnight, and PBS washes 3 × 3min；6. life
The anti-sheep IgG1 of thing elementization pig:200,37 DEG C, 30min；7.PBS washes 3 × 3min；8.Streptavidin-HRP, 1:200 37 DEG C,
30min；9.PBS washes 3 × 3min；
10.0.05%DAB+0.03%H2O2Develop the color 8~12min, washing；11. haematoxylin lining dye, washing；12. drying, tree
Fat mounting；13. result is observed：The positive is in sepia, and background is hyacinthine.As a result such as Fig. 4.
2. Toluidine blue staining detects the content and the content of cartilage matrix mucopolysaccharide (GAG) of nucleus in cartilage：
1st, paraffin section de-waxing is to water；2nd, distilled water embathes 3 times；3rd, 0.1% toluidine blue immersion dye 10min；4th, wash,
Wash away unnecessary dye liquor；5th, dehydration of alcohols at different levels；6th, dimethylbenzene is transparent, neutral gum sealing.As a result such as Fig. 5
3. HE dyeing detects the content of nucleus in cartilage：
1st, paraffin section de-waxing is to water：Section is put into the 20min- absolute ethyl alcohols I of I 20min- dimethylbenzene of dimethylbenzene II successively
The 10min-95% alcohol 5min-90% alcohol 5min-80% alcohol 5min-70% alcohol 5min- of 10min- absolute ethyl alcohols II steams
Distilled water is washed.2nd, bush uniformly dyeing nucleus：Cut into slices into Harris bush uniformly dyeing 3-8min, originally wash, 1% hydrochloride alcohol differentiation
Several seconds, running water is rinsed, and 0.6% ammoniacal liquor returns indigo plant, and flowing water is rinsed.3rd, Yihong dye cytoplasm：Cut into slices and dye 1- in eosin stain
3min.4th, it is dehydrated mounting：By section be sequentially placed into the 5min- of 95% alcohol I5min-95% alcohol II 5min- absolute ethyl alcohols I without
It is dehydrated transparent in the 5min of II 5min- dimethylbenzene of water-ethanol, I 5min- dimethylbenzene II, section is taken out from dimethylbenzene and slightly dried, in
Property gummy mounting.As a result such as Fig. 6.
Embodiment 3 takes off cell processing
Cartilage row is handled as follows:1. cartilage is inserted in Tris-HCl (pH7.4) buffer solution, buffer solution inner protein enzyme
Inhibitor (mass fraction be 0.035% PMSF), 4 DEG C of isothermal vibrations 48 hours；2. 2%SDS hypotonic Tris-HCl are buffered
Add protease inhibitors (mass fraction is 0.035% PMSF) in solution (volume/volume), 4 DEG C of isothermal vibrations are after 48 hours
Take out, with distilled water continuous flushing 24 hours；3. 37 DEG C of digestion 4 of 1g/LRnaseA, 500U/mLDnase I mixture slakings enzyme liquid
Hour；4. in the hypotonic Tris-HCl cushioning liquid for inserting the X-100 containing 1%Triton, 4 DEG C digest 24 hours, are distilled after taking-up
Water continuous flushing 48 hours, completes to handle the de- cell of cartilage, becomes cartilage acellular matrix (Cartilage
Acellular Matrix；CACM).
1. in II Collagen Type VIs SABC detection cartilage II Collagen Type VIs situation, method be the same as Example 2, as a result such as Fig. 7, not
De- cytohyalop lasm cartilage and de- cytohyalop lasm cartilage carry out II Collagen Type VIs, as can be seen that not taking off cell from experimental result
The II Collagen Type VIs content of hyalomitome cartilage and de- cytohyalop lasm cartilage almost, but is seen in de- groups of cells inside cartilage cavities
Less than the presence of nucleus, and de- groups of cells can significantly see the presence of nucleus, that is, take off the hyalomitome cartilage of groups of cells
Cell is taken off.
2. Toluidine blue staining detects the content of nucleus in cartilage, method be the same as Example 2, as a result such as Fig. 8；From toluidines
Indigo plant dyeing is as can be seen that not cell free hyalomitome cartilage is more than de- groups of cells toluidine blue content, but de- groups of cells is soft
The presence of nucleus is can't see in bone lacuna, i.e., acellular presence, the cell represented in hyalomitome cartilage is taken off.
3. HE dyeing detects the content of nucleus in cartilage, method be the same as Example 2, as a result such as Fig. 9；
From HE dyeing as can be seen that seeing the presence it can be seen that nucleus in not cell free hyalomitome cartilage cavities, and
The presence that cell free hyalomitome cartilage is acellular, the cell represented in hyalomitome cartilage is taken off.
4. by the de- cytohyalop lasm cartilage matrix (ECM) (n=3) of preparation and not cell free hyalomitome cartilage (n=
3), dissolved through 50mM sodium citrate solutions, 2000r/min, centrifuge 10min, abandon supernatant, add ethylenediamine tetra-acetic acid and pawpaw egg
White enzyme is cracked 72 hours in 65 DEG C of water-baths, and 10000r/min, 5min centrifuging and taking supernatants, sample adds equivalent
Hoechst33258, the DNA extracted using calf thymus are as standard items, DNA content in fluorescence colorimetric assay detection sample, such as
Figure 10, table 1.
Comparative example 1
Pig knee cartilage is rinsed repeatedly, and puts in pulverizer and is crushed, it is 4 to be then placed in pH7.5, temperature
Soaked in DEG C sterile PBS, centrifuge 5min, speed 2000rpm, remove precipitation.Upper strata cartilage fragment is subjected to 5min centrifugations again,
Speed is 8000rpm, filters out cartilage grain of the diameter at 100-154 μm, is carried out according to improved Courtman improved methods de- thin
Born of the same parents are handled.1. it is 0.25% trypsase and 0.03%EDTA PBS to be added into the cartilage grain of acquisition containing concentration, shakes 1h,
Centrifugation 3 times, speed is 7 000rpm, is rinsed using PBS.2. at 4 DEG C add concentration for 1% Triton X-100 and
10mM Tris-HCl (pH=7.5) and concentration is 0.035% PMSF, shakes 24-72h, is rinsed after centrifugation using PBS.③
Concentration is put at 37 DEG C for 50U/mLDNA enzymes and 1U/mL RNases, 12h is digested, is rinsed after centrifugation using PBS.4. in temperature
Tris-HCl (pH=7.5) of the concentration for 1% Triton X-100 10mM is added at 4 DEG C, is obtained after concussion 24h, flushing
White powder is precipitated.Put it into grinding tool, acellular matrix is obtained after freezing, drying.
1. in II Collagen Type VIs SABC detection cartilage II Collagen Type VIs situation, method be the same as Example 2, as a result such as Figure 11,
Natural cartilage acellular matrix and tissue engineering bone/cartilage acellular matrix carry out II Collagen Type VI detections, can from experimental result
Go out, natural cartilage acellular matrix II Collagen Type VIs content more cell free than tissue engineering bone/cartilage is few, and tissue engineering bone/cartilage is de-
The presence of nucleus is can't see inside cartilage cavities in cellular matrix, and natural cartilage acellular matrix group can significantly be seen
It is more easy to take off cell than hyalomitome cartilage to the presence of nucleus, i.e. tissue engineering bone/cartilage.
2. Toluidine blue staining detects the content of nucleus in cartilage
The content of nucleus, method be the same as Example 2, as a result such as Figure 12 in Toluidine blue staining detection cartilage；From toluidines
Indigo plant dyeing is as can be seen that the GAG contents of tissue engineering bone/cartilage acellular matrix kind are than the content in natural cartilage acellular matrix
It is many.And the presence of nucleus is can't see inside the cartilage cavities in tissue engineering bone/cartilage acellular matrix, and natural cartilage is de- thin
Cytoplasmic matrix group can significantly see that the presence of nucleus, i.e. tissue engineering bone/cartilage are more easy to take off cell than hyalomitome cartilage.
3. HE dyeing detects the content of nucleus in cartilage
The content of nucleus, method be the same as Example 2, as a result such as Figure 13 in Toluidine blue staining detection cartilage；From HE dyeing
As a result find out in, the presence of nucleus is can't see inside the cartilage cavities in tissue engineering bone/cartilage acellular matrix, and it is natural soft
Bone acellular matrix group can significantly see the presence of nucleus, that is, further illustrate tissue engineering bone/cartilage than hyalomitome cartilage
It is more easy to take off cell.
4. DNA content is determined
The content of nucleus (or DNA content) in DNA content measure detection cartilage, method be the same as Example 2, as a result such as
Figure 10, table 1.As can be seen from the results, compared with tissue engineering bone/cartilage acellular matrix, contain in natural cartilage acellular matrix
More DNA.That is the de- cell of natural cartilage is not thorough.
The cartilage cell's pellet culture method of comparative example 2 (conventional organization engineered cartilage cultural method)
1st, cartilage cell is digested
Primary swine chondrocytes culture was to the 8th day, and cartilage cell's degrees of fusion reaches 95%.Trypsase is placed in 37 DEG C in advance
Preheating, digests cartilage cell, 0.25% Trypsin Induced 4min.With CM culture mediums and trypsase 1：1 ratio, which is terminated, to disappear
Change.Cell is counted with tally, 1100rpm, 5min centrifuge cells, removes supernatant.
2nd, cartilage cell's pellet culture
Take 3 × 105Individual/in conical pipe, 120g centrifugations cultivate (uncapping, be placed in incubator) in culture medium.2
After it, agglomerate is transferred in not coated 24 orifice plate, further breaks up it.
Wherein culture medium is DMEM/F12, and 1% is dual anti-, 1%NEAA, 1% (ITS), 1% vitamin C, 10ng/ml TGF-
β3.Culture was to the 20th day, and cell mass forms tissue.
3rd, cell processing is taken off
Experimental procedure such as the present embodiment 3.
Test effect：
The measure of the cartilage DNA content of table 1
Data are from the tissue engineering bone/cartilage prepared with embodiment 2 in table 1, the de- cell processing through embodiment 3；It is natural soft
The pig natural cartilage that bone is obtained from embodiment 1；Cell mass cultural method of the conventional organization engineered cartilage in comparative example 2
The tissue engineering bone/cartilage of acquisition.
As a result as can be seen that do not take off groups of cells (untreated) tissue engineering bone/cartilage can detect DNA (100%,
Concentration is 400ng/mg).And the tissue engineering bone/cartilage of de- groups of cells and be not detected by DNA presence (0%, concentration is 0ng/
Mg), i.e., in the absence of cell.Conventional organization engineered cartilage detects DNA remnants 37.5% after taking off cell, and content is far longer than this reality
Apply the tissue engineering bone/cartilage acellular matrix DNA content in example 2.The more conventional tissue of tissue engineering bone/cartilage i.e. in the present embodiment 2
Engineered cartilage is easy to de- cell processing.
Also, tissue engineering bone/cartilage institutional framework prepared by embodiment 3 is good, and cell distribution is uniform.With natural cartilage phase
Than spongiform rarefaction is presented in the tissue engineering bone/cartilage of embodiment 3.Can well it be contacted with the cartilaginous tissue of surrounding,
And cell is easy to slough；And the cartilage of dimension can be cut into as needed；Importantly, made from embodiment 3
It is hyalomitome cartilage, the II Collagen Type VIs content of secretion is high, that is, primarily forms hyalomitome cartilage (composition is similar to natural cartilage).And
Then cell arrangement is close for conventional organization engineered cartilage (comparative example 2), and cell is difficult to slough.It is in irregular shape, with surrounding tissue not
Contact well can be formed.Type i collagen content in agglomerate is high, that is, forms the higher fibrocartilage of content.Compare with embodiment 3
Compared with, it is slower with the cell growth that same cell inoculum concentration, identical incubation time are carried out in dimensional culture, comparative example 2, formation
Agglomerate is smaller.
It the above is only the preferred embodiment of the present invention, it is noted that come for those skilled in the art
Say, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (10)
1. a kind of preparation method of cartilage acellular matrix, it is characterised in that by tissue engineering bone/cartilage successively through A liquid, the processing of B liquid
Afterwards, digested, then handled through C liquid with RnaseA and Dnase I, obtain de- cellular cartilage；
The A liquid is the Tris-HCl buffer solutions containing protease inhibitors, and the mass fraction of wherein protease inhibitors is
0.01%~1%；
The B liquid is the mass fraction of Tris-HCl buffer solutions, wherein protease inhibitors containing SDS and protease inhibitors
For 0.01%~1%；SDS volume fraction is 2%；
The C liquid is the Tris-HCl buffer solutions containing Triton X-100, and wherein Triton X-100 volume fraction is
1%.
2. preparation method according to claim 1, it is characterised in that the preparation method of the tissue engineering bone/cartilage is：
Step 1：After autologous cartilage is digested with II Collagen Type VIs, original cuiture is carried out with CM culture mediums；
Step 2：After cell after original cuiture is with Trypsin Induced, it is resuspended with sodium alginate soln；
Step 3：The cell of resuspension is connected to the coated culture vessel of gelatin, 0~4 DEG C is placed after 4min, adds CaCl2Solution, 0
~4 DEG C of placement 4min, form Cellular gels；
Step 4：Cellular gels are transferred to the coated culture vessel of agarose, with CC medium cultures 15~25 days, cultivated
Change fresh culture within every 2~3 days in journey；Obtain tissue engineering bone/cartilage.
3. preparation method according to claim 2, it is characterised in that the matter of the middle sodium alginate of the sodium alginate soln
It is 0.1%~15% to measure fraction.
4. preparation method according to claim 2, it is characterised in that the middle cell of the resuspension to sodium alginate soln
Density is 2 × 106Individual cell/mL.
5. preparation method according to claim 2, it is characterised in that the CaCl2The concentration of solution is 200mmol/L；
CaCl2The volume ratio of solution and sodium alginate soln is 1：1.
6. preparation method according to claim 1, it is characterised in that the condition of the A liquid processing is 0~4 DEG C, at concussion
Manage 48h.
7. preparation method according to claim 1, it is characterised in that the condition of the B liquid processing is 0~4 DEG C, at concussion
48h is managed, then with distilled water flushing 24h.
8. preparation method according to claim 1, it is characterised in that in RnaseA the and Dnase I enzymolysis enzymolysis,
RnaseA amount is 1g/L；Dnase I amount is 500U/mL.
9. preparation method according to claim 1, it is characterised in that the condition of the C liquid processing is 0~4 DEG C, digestion
24h, then with distilled water flushing 24h.
10. cartilage acellular matrix made from any one of claim 1~9 preparation method.
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