CN106267355A - A kind of method promoting chondrocyte to stick and the tissue engineering bone/cartilage of structure thereof - Google Patents
A kind of method promoting chondrocyte to stick and the tissue engineering bone/cartilage of structure thereof Download PDFInfo
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- CN106267355A CN106267355A CN201610891557.8A CN201610891557A CN106267355A CN 106267355 A CN106267355 A CN 106267355A CN 201610891557 A CN201610891557 A CN 201610891557A CN 106267355 A CN106267355 A CN 106267355A
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- chondrocyte
- cartilage
- avidin
- stick
- tissue engineering
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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
- A61L27/38—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 containing added animal cells
- A61L27/3804—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 containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
-
- 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
- A61L27/38—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 containing added animal cells
- A61L27/3839—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 containing added animal cells characterised by the site of application in the body
- A61L27/3843—Connective tissue
- A61L27/3852—Cartilage, 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
- 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
- A61L27/38—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 containing added animal cells
- A61L27/3895—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 containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
-
- 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
Abstract
The invention discloses a kind of method promoting chondrocyte to stick and the tissue engineering bone/cartilage of structure thereof.The method is by by the monoclonal antibody of chondrocyte surface high expressed antigen and biotin-labeled pentylamine system phase coupling, avoid the chondrocyte endocytosis problem to biotin in biotin-labeled pentylamine system, on the other hand the adhesion that antigen-antibody is powerful is utilized, increase chondrocyte Adhering capacity on biomaterial, promote propagation and the expression of chondrocyte phenotype of Seed Cells of Tissue Engineering Cartilage, and then build the repairing effect of tissue engineering bone/cartilage raising cartilage defect.
Description
Technical field
The present invention relates to a kind of method promoting chondrocyte to stick and the tissue engineering bone/cartilage of structure thereof.
Background technology
The reparation of articular cartilage defect is the difficult point of Orthopedic Clinical.The rise of tissue engineering technique carries for solving this problem
Supply new dawn.But its repairing effect and normal cartilage still have the biggest gap.Thinking, seed cell and support carry
Body adhesive force is not strong, and seed cell comes off from support, causes seed cell lazy weight to be probably and causes this undesirable effect
The one of the main reasons of fruit.In biological tissue, cell and sticking of extracellular matrix are by receptor on cell membrane and cell
The combination of the adhesion protein in epimatrix completes.Obviously, tissue engineering bracket material lacks this type of adhesion protein.Therefore, carefully
Born of the same parents can only be adhered on it by the effect such as ionic attraction, hydrogen bonded.Therefore the Adhering capacity that seed cell is on artificial material is very
Difference.And organizational project frequently with several main seed cell in, the Adhering capacity of chondrocyte is worst.Biotin
(biotin, B)-Avidin (avidin, A) technology (hereinafter referred to as BA technology) is a development in recent years biology rapidly
Technology, and have been obtained for extensively applying.BA technological system can improve chondrocyte Adhering capacity, and does not change chondrocyte
Gene expression pattern, its know-why is as shown in Figure 1.It is applied to couple endotheliocyte and artificial blood by BA technology at Chan etc.
After tube material, the adhesive force of endotheliocyte significantly increases, and cell number bound on support improves 7.5 times.BA technology is bound
On the transmission capacity of its function and transmembrane signal almost without impact after hepatocyte.But along with the prolongation of incubation time, chondrocyte
The biotin that the endocytosis of biotin result in its surface fades away, and have impact on BA technological system long-term role.
Cultivation temperature is dropped to 4 DEG C of chondrocyte endocytosiss to biotin of can slowing down, but significantly reduces chondrocyte
Existence and multiplication capacity.Therefore the endocytosis problem of biotin is still had to be solved by chondrocyte.
Summary of the invention
Because the drawbacks described above of prior art, the invention provides a kind of method promoting chondrocyte to stick, the party
Method is by by the monoclonal antibody of chondrocyte surface high expressed antigen and biotin-avidin system phase coupling, it is to avoid life
The chondrocyte endocytosis problem to biotin in thing element-Avidin system, on the other hand utilizes the adhesion that antigen-antibody is powerful,
Increase chondrocyte Adhering capacity on biomaterial, promote propagation and the chondrocyte table of Seed Cells of Tissue Engineering Cartilage
The expression of type, and then build the repairing effect of tissue engineering bone/cartilage raising cartilage defect.
Further, the method that promotion chondrocyte of the present invention sticks includes step:
1) by biomaterial Avidin；
2) monoclonal antibody of chondrocyte with biotinylated chondrocyte surface high expressed antigen is combined；
3) by step 1) in biomaterial and the step 2 of Avidin that prepare) in prepared to be combined with biotinylation soft
The chondrocyte of the monoclonal antibody of osteocyte surface high expressed antigen passes through biotin-avidin system phase coupling.
Said method by biotin (biotin, B)-Avidin (avidin, A) technology (hereinafter referred to as BA technology) system and
The antibody of chondrocyte surface high expressed antigen couples mutually, and BA technological system is improved to monoclonal antibody-BA technological system (i.e.
Monoclonal antibody-Biotin-Avidin technological system, as a example by monoclonal antibody CD44, hereinafter referred to as CBA technology, the schematic diagram of the inventive method
As shown in Figure 2).
Preferably, step 1) concrete operations for biomaterial is immersed in Avidin solution, during oscillating reactions one section
Between, make both combine；Wherein the response time is preferably 0.5～2 hour, and the concentration of Avidin solution is preferably 0.05～0.2mg/
mL。
Further, step 1) described biomaterial is the material being combined with Avidin, preferably porous support is biological
Material, such as chitosan porous rack, and the porosity of described porous support is preferably 80～95%, and aperture is preferably 100～150
μm。
In the preferred embodiment of the present invention, described biomaterial is cylinder chitosan porous rack, this
A diameter of the 8 of frame～15mm, height is 2～5mm.
Preferably, step 2) concrete operations be by chondrocyte and biotinylation chondrocyte surface high expressed antigen
Monoclonal antibody standing and reacting, and the usage amount of the monoclonal antibody of described biotinylation chondrocyte surface high expressed antigen is
0.2～0.3 μ g/ million chondrocyte；The time of described standing and reacting is preferably 20～40 minutes, most preferably 30 minutes.
Further, described chondrocyte surface high expressed antigen preferably is selected from CD44, CD151 and CD29.
Preferably, step 3) concrete operations be in the biomaterial of Avidin inoculation be combined with biotinylation cartilage
The chondrocyte suspension of the monoclonal antibody of cell surface high expressed antigen, is statically placed in cell culture incubator, inoculates a period of time
Rear replacing culture fluid continues to cultivate, and prepares tissue engineering bone/cartilage.
Preferably, the condition of culture of described cell culture incubator is 37 DEG C, 5%CO2。
Present invention also offers the monoclonal antibody by chondrocyte surface high expressed antigen and biotin-avidin system
System phase coupling and make chondrocyte attach to the tissue engineering bone/cartilage constructed by biomaterial surface.
Further, monoclonal antibody one end of described chondrocyte surface high expressed antigen combines chondrocyte, the other end
Couple biotin-Avidin system, and described biotin-avidin system combines with biomaterial.
Preferably, described biomaterial is porous support.
The method that promotion chondrocyte of the present invention sticks can be used for the preparation of tissue engineering bone/cartilage, and it is prepared
Tissue engineering bone/cartilage can be used for repairing and other relevant orthopedics clinic practice of articular cartilage defect.
The method that promotion chondrocyte of the present invention sticks, on the one hand, utilize BA technological system to divide as bridge, two ends
Bang Ding biomaterial and antibody, it is to avoid biotin directly contacts with chondrocyte, thus solves chondrocyte to life
The endocytosis problem of thing element, on the other hand, utilizes the adhesion that antigen-antibody is powerful, it is ensured that BA technological system is for chondrocyte
Binding, thus increase chondrocyte Adhering capacity on biomaterial.
Below with reference to accompanying drawing, the technique effect of design, concrete structure and the generation of the present invention is described further, with
It is fully understood from the purpose of the present invention, feature and effect.
Accompanying drawing explanation
Fig. 1 is BA technical schematic diagram；
Fig. 2 is CBA technical schematic diagram.
Detailed description of the invention
Embodiment 1 swine chondrocytes separation and Culture
Separating small pig knee joint under aseptic condition, and remove the soft tissues such as knee joint week muscle, tendon and ligament.Rinse cartilage
Surface is to without obvious knuckle synovia.Interior outside condyle of femur cartilage flake is cut down by knife blade, notes only collecting cartilage layers, extremely
When there is petechial hemorrhage on surface, stop cutting.Cartilage sheet is put into culture dish, immerses and DMEM rinses for several times, shred to 1mm ×
1mm size, DMEM rinses again, after removing supernatant moves into cartilaginous tissue equipped with 250mL Collagenase Type Han 0.2%II
The DMEM in high glucose solution of (Collagenase Type II, Sigma company, the U.S.), be transferred to 37 DEG C of isothermal vibration casees (Sanyo,
Japan) 4-6h, every 2h note observe cartilage digestion situation, without shake again after clearly visible cartilage grain 0.5h with ensure disappear completely
Change.After having digested, Digestive system is transferred in super-clean bench (Sanyo, Scb-120, Japan) operation.With 70 μm cell filters
Filter, be distributed into 6 50mL centrifuge tubes centrifugal (1500rpm × 5min).By each solencyte 10mL PBS suspendible, arrange to 2
Individual 50mL centrifuge tube, recentrifuge (1500rpm × 5min) after leveling.Obtained cell with cultured chondrocytes base (containing 10%FBS
DMEM, 4500mg/L glucose, 584mg/L glutamine, 100U/mL penicillin, 100 μ g/mL streptomycins, 10mM
The nonessential aminoacid of HEPES, 0.1mM, 0.4mM proline, 50mg/L ascorbic acid) suspendible again, cell counter counts, presses
5.0×105/ mL inoculates into 75cm2Culture bottle, cultivate in cell culture incubator (Sanyo, MCA-152C, Japan) (37 DEG C, 5%
CO2).Unnecessary cell presses 5.0 × 106/ mL is frozen.48h backsight cell attachment situation changes culture fluid, often within 2-3 days, changes liquid later
1 time.Observation of cell growing state under inverted microscope (Olympus, TH4-100, Tokyo, Japan).When seeing under inverted microscope
Examine chondrocyte merge into sheet cover with major part culture bottle wall to 90% merge after, incline culture fluid, add 0.025% (w/v)
Trypsin+0.01% (w/v) ethylenediaminetetraacetic acid digestion 5-8min, collects cell by 5.0 × 105/ mL carries out Secondary Culture.
This part institute cell is second filial generation swine chondrocytes.
Embodiment 2 chitosan stent Avidin
Chitosan multi-porous three-dimensional rack is prefabricated into diameter 10mm, the big small cylinder of high 3mm, porosity ＞ 90%, aperture
100～150 μm, ethylene oxide sterilizing is standby.Take above-mentioned chitosan stent and put in 12 well culture plates, add 0.1mg/mL Avidin
Solution 3.0mL, (125r/min) reaction 1h on shaking table under room temperature, with dual anti-containing 200mg/mL and 25mg/mL amphotericin B
PBS washs 2 times, standby after natural drying.
Embodiment 3 is by CD44 monoclonal antibody-biotin-avidin system external structure tissue engineering bone/cartilage
Chondrocyte is with 5 × 107After individual/mL density and biotinylation monoclonal antibody CD44 standing and reacting 30min, (0.25 μ g is biological
Elementization monoclonal antibody CD44/1 × 106Individual cell) PBS wash 2 times, be inoculated in place chitosan stent 24 orifice plates, each support
Middle inoculation 200 μ L cell suspension, is statically placed in 37 DEG C, 5%CO2Incubator cultivate, make cell and support further combined with.After inoculation 4h
Transfer cytoskeleton complex to 24 new orifice plates, gently add 2mL culture fluid stand incubator cultivate, the next day change liquid.
The work process of 4 products of embodiment.
Repair pig knee joint cartilages in loaded parts full-thickness defects: Experimental Miniature Pig male and female do not limit, 3 monthly ages, body weight 15 ±
2.5kg.All animals are all without field of operation wound, infection, deformity and other general diseases.Ketamine 10mg/kg intramuscular injection induction fiber crops
Liquor-saturated, Su Mian Xin 0.1mL/kg intramuscular injection is anaesthetized, outside longitudinal incision before double knee joint, and patella pulls to inner side, exposes outside knee joint femoral
Condyle heavy burden district, uses trepan to produce the cylindric Complete cartilage Defect of diameter 10mm, deep 3mm.Application CD44 monoclonal anti
Body-biotin-avidin system external structure tissue engineering bone/cartilage repairs pig knee joint cartilages in loaded parts full-thickness defects.During operation
The a small amount of medical adhesive of cartilage defect bottom spray, closely inlays implantation cartilage defect by diameter 10mm, high 3mm tissue engineering bone/cartilage
Place, thoroughly stops blooding, the most tightly sews up the incision and close wound with medical adhesive.In art and postoperative 1 day every pig to give celebrating the most mould
Element 240,000 U intramuscular injection prevention is infected, and postoperative is allowed to free activity.Within postoperative 24 weeks, put to death animal, cut whole knee joint, demonstrate,prove after detection
Real cartilage defect repair is good.
The preferred embodiment of the present invention described in detail above.Should be appreciated that those of ordinary skill in the art without
Need creative work just can make many modifications and variations according to the design of the present invention.Therefore, all technology in the art
Personnel are available by logical analysis, reasoning, or a limited experiment the most on the basis of existing technology
Technical scheme, all should be in the protection domain being defined in the patent claims.
Claims (10)
1. the method promoting chondrocyte to stick, it is characterised in that described method is by by chondrocyte surface overexpression protection
Former monoclonal antibody and biotin-avidin system phase coupling.
2. the as claimed in claim 1 method promoting chondrocyte to stick, it is characterised in that include step:
1) by biomaterial Avidin；
2) monoclonal antibody of chondrocyte with biotinylated chondrocyte surface high expressed antigen is combined；
3) by step 1) in biomaterial and the step 2 of Avidin that prepare) in prepared to be combined with biotinylation cartilage thin
The chondrocyte of the monoclonal antibody of cellular surface high expressed antigen passes through biotin-avidin system phase coupling.
3. the as claimed in claim 2 method promoting chondrocyte to stick, it is characterised in that step 1) concrete operations for will
Biomaterial is immersed in Avidin solution, oscillating reactions a period of time, makes both combine.
4. the as claimed in claim 2 method promoting chondrocyte to stick, it is characterised in that step 1) described biomaterial is
The porous support being combined with Avidin.
5. the as claimed in claim 2 method promoting chondrocyte to stick, it is characterised in that step 2) concrete operations for will
Chondrocyte and the monoclonal antibody standing and reacting of biotinylation chondrocyte surface high expressed antigen.
6. the as claimed in claim 2 method promoting chondrocyte to stick, it is characterised in that step 3) concrete operations be
In the biomaterial of Avidin, inoculation is combined with the soft of the monoclonal antibody of biotinylation chondrocyte surface high expressed antigen
Osteocyte suspension, is statically placed in cell culture incubator, changes culture fluid and continues to cultivate, prepare organizational project soft after inoculation a period of time
Bone.
7. the tissue engineering bone/cartilage that the method sticked with the promotion chondrocyte as described in claim 2-6 builds.
8. the method that the promotion chondrocyte as described in claim 2-6 sticks application in prepared by tissue engineering bone/cartilage.
9. a tissue engineering bone/cartilage, it is characterised in that it is that the monoclonal antibody by chondrocyte surface high expressed is with biological
Element-Avidin system phase coupling and make chondrocyte attach to biomaterial surface.
10. tissue engineering bone/cartilage application in Articular cartilage repair as claimed in claim 9.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058217A (en) * | 2017-01-22 | 2017-08-18 | 广州赛莱拉干细胞科技股份有限公司 | The proliferated culture medium and Multiplying culture method of a kind of cartilage cell |
| CN113234669A (en) * | 2021-05-10 | 2021-08-10 | 深圳市第二人民医院(深圳市转化医学研究院) | Culture solution for delaying dedifferentiation of chondrocytes and preparation method thereof |
Citations (2)
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|---|---|---|---|---|
| CN102176882A (en) * | 2008-08-07 | 2011-09-07 | 生物活性外科公司 | Stem cell capture and immobilization coatings for medical devices and implants |
| CN103599569A (en) * | 2013-11-27 | 2014-02-26 | 天津大学 | Preparation method of titanium alloy surface growth factor composite coating |
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2016
- 2016-10-12 CN CN201610891557.8A patent/CN106267355A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102176882A (en) * | 2008-08-07 | 2011-09-07 | 生物活性外科公司 | Stem cell capture and immobilization coatings for medical devices and implants |
| CN103599569A (en) * | 2013-11-27 | 2014-02-26 | 天津大学 | Preparation method of titanium alloy surface growth factor composite coating |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058217A (en) * | 2017-01-22 | 2017-08-18 | 广州赛莱拉干细胞科技股份有限公司 | The proliferated culture medium and Multiplying culture method of a kind of cartilage cell |
| CN113234669A (en) * | 2021-05-10 | 2021-08-10 | 深圳市第二人民医院(深圳市转化医学研究院) | Culture solution for delaying dedifferentiation of chondrocytes and preparation method thereof |
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Application publication date: 20170104 |