CN108096632A - Articular cartilage repair materials and preparation method based on oxidized hyaluronic acid-II Collagen Type VIs and self concentration bone marrow nucleated cell - Google Patents
Articular cartilage repair materials and preparation method based on oxidized hyaluronic acid-II Collagen Type VIs and self concentration bone marrow nucleated cell Download PDFInfo
- Publication number
- CN108096632A CN108096632A CN201711446501.2A CN201711446501A CN108096632A CN 108096632 A CN108096632 A CN 108096632A CN 201711446501 A CN201711446501 A CN 201711446501A CN 108096632 A CN108096632 A CN 108096632A
- Authority
- CN
- China
- Prior art keywords
- hyaluronic acid
- bone marrow
- self
- oxidized hyaluronic
- nucleated cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
-
- 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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0669—Bone marrow stromal cells; Whole bone marrow
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Dermatology (AREA)
- Transplantation (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- General Chemical & Material Sciences (AREA)
- Vascular Medicine (AREA)
- Developmental Biology & Embryology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Rheumatology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Materials For Medical Uses (AREA)
Abstract
The present invention relates to a kind of articular cartilage repair materials and preparation method based on oxidized hyaluronic acid II Collagen Type VIs and self concentration bone marrow nucleated cell, by oxidized hyaluronic acid, II collagen types and self concentration bone marrow nucleated cell are made, self concentration bone marrow nucleated cell in material has self-renewing and into cartilage differentiation potential, participate in cartilage defect repair, therefore with good cartilage damage repairing effect, the transparent cartilage sample of repair tissue, repair tissue surface smoothness, with the degree of integration of neighbouring normal cartilage, II collagen type contents, GAG contents, calcified cartilage layer and subchondral bone form etc. are significantly better than that blank control group, there is the clinical potential converted.
Description
Technical field
The invention belongs to medical material fields, and being related to is had based on oxidized hyaluronic acid-II Collagen Type VIs and self concentration marrow
The articular cartilage repair materials of nucleus further relate to the preparation method and application of the material.
Background technology
Articular cartilage is the thin layer dense connective tissue that lining is attached to articular surface, and surface is smooth, and quality is firm and rich
Elasticity is the important heavy burden tissue of human synovial.And normal articular cartilage is damaged since no blood vessel, nerve and lymph are distributed
After be difficult to self-healing, still lack effective correcting strategy so far.And in clinical position, the incidence of articular cartilage damage compared with
Height, 25,124 laparoscopic surgery cases of Widuchowski retrospective analysis find that there are cartilage damages for 60% case.It is soft
Bone injury, which has become, causes arthralgia, the dysfunction even major reason of physical disabilities.Clinically used damage at this stage
Repair of cartilage strategy mainly includes：Articular cavity cleaning, micro fractures, cartilage transplantation, chondrocyte cell transplantation, bone cartilage transplantation etc., but
More than correcting strategy long-term efficacy is not good enough, and therapeutic effect is unsatisfactory.Cartilage tissue engineered technology is articular cartilage damage
Repair ideal method, but at this stage tissue engineering technique for repair of cartilage there are still problems, such as：Program is more multiple
It is miscellaneous；Repair tissue mechanical attribute is poor, integrates not good enough and fibrocartilage regression etc. with host tissue.Therefore, explore more simple
Just and the cartilage tissue engineered strategy with more excellent repairing effect is of great significance.
The content of the invention
In view of this, one of the objects of the present invention is to provide a kind of based on oxidized hyaluronic acid-II Collagen Type VIs and self
Concentrate the articular cartilage repair materials of bone marrow nucleated cell；The second object of the present invention is to provide based on oxidized hyaluronic acid-
The preparation method of the articular cartilage repair materials of II Collagen Type VIs and self concentration bone marrow nucleated cell；The third object of the present invention exists
In answering for articular cartilage repair materials of the offer based on oxidized hyaluronic acid-II Collagen Type VIs and self concentration bone marrow nucleated cell
With.
In order to achieve the above objectives, the present invention provides following technical solution：
1. the articular cartilage repair materials based on oxidized hyaluronic acid-II Collagen Type VIs and self concentration bone marrow nucleated cell,
It is made of oxidized hyaluronic acid, II collagen types and self concentration bone marrow nucleated cell.
Preferably, the addition of oxidized hyaluronic acid, II collagen types and self concentration bone marrow nucleated cell by every 5 ×
106A self concentration bone marrow nucleated cell adds in 100mg II collagen types and is uniformly mixed, and is shaken and centrifuged by whirlpool, gone
It, will be in the II collagen types of 10ul oxidized hyaluronic acid solution addition compound cells, gel except bubble in mixed gel.
Preferably, the oxidized hyaluronic acid of the 100mg/ml is molten by every 100mg oxidized hyaluronic acid addition 1ml PBS
Liquid, it is obtained through being completely dissolved.
It is furthermore preferred that the II collagen types are prepared by following methods：It is 20 that lyophilized gartilage powder is pressed mass volume ratio:
1(g：L it is) that the stirring of 4M guanidine hydrochloride solutions is suspended with concentration, stirring is placed under the conditions of 4 DEG C, is digested viscous more in gartilage powder matrix
Sugar extremely homogenate state；After digestion completely, digestion product is centrifuged into 20min under the conditions of 4 DEG C, 8000rpm, abandons supernatant, collected
Sediment is dissolved in water, and then adds in the acetic acid for being equivalent to 3/50 times of guanidine hydrochloride solution volume, and addition is equivalent to lyophilized gartilage powder
The pepsin that 0.1 times of weight adds water to final volume as 2 times of guanidine hydrochloride solution, pH value is adjusted between 2.5-3, then 4
48h is digested under the conditions of DEG C；Digestion is finished centrifuges 20min after 8000rpm in vertical high speed centrifugal machine, abandons precipitation, collects supernatant
Liquid；Then it is poured slowly into supernatant when the NaOH solution that concentration is 1.5M and stirring to pH value are 7.5 and stops falling to note, is added in
NaCl makes its final concentration of 3M, and under the conditions of 4 DEG C, 8000rpm centrifugation 20min collect sediment；Sediment is dispensed into bag filter
Treat conductivity of dialysate, close to pure water conductivity when terminates to dialyse；By sticky colloidal solution in bag filter under the conditions of 4 DEG C, stand
Formula supercentrifuge 8000rpm centrifuges 20min, collects sediment into sterile volumetric flask, the hydrochloric acid for adding in 0.15mol/L is molten
Liquid, white precipitate albumen disperses, dissolves in gelatinous after dissolving 3 days, stirs evenly, and dispenses, and freezes, spare.
It is furthermore preferred that the oxidized hyaluronic acid is prepared by following methods：It is configured to the hyaluronic acid that mass fraction is 2%
Solution, then adds in the sodium periodate solution of 0.1g/ml, and stirring when complete oxidation 6 is small under the conditions of being protected from light, then adds in
Ethylene glycol terminates reaction, then when reaction solution is poured into bag filter to electrical conductivity close to pure water terminates to dialyse, and it is transparent to obtain oxidation
Matter acid.
It is furthermore preferred that concentration bone marrow nucleated cell is prepared by following methods self：Current animal is anaesthetized, Ran Houyong
Then the syringe bone marrow extraction blood prewetted using 1000U/ml heparin is added in the anti-freezing bone marrow fluid of extraction in equal volume
Sterile PBS liquid, mixing, it is 1.073g/ml's to be slowly injected into bone marrow fluid containing isometric concentration along tube wall after mixing
In Percoll born of the same parents' separating liquid, interface is complete between injection process holding separating liquid and mixing liquid, then using Percoll
Density-gradient centrifugation method obtains self concentration bone marrow nucleated cell, first centrifuges mixing liquid 15min with the speed of 2000rpm, inhales
Karyocyte layer is taken, the karyocyte that PBS solution washing is drawn is added in, with the speed centrifuge washing cell 5min of 1500rpm, abandons
Supernatant.Add in PBS piping and druming washing cells again, 1000rpm speed centrifugation 5min abandons supernatant, adds in PBS and be resuspended to obtain the final product.
2nd, the articular cartilage based on oxidized hyaluronic acid-II Collagen Type VIs and self concentration bone marrow nucleated cell repairs material
The preparation method of material, is as follows：Bone marrow nucleated cell will be concentrated self first to mix with II collagen types, then be added again
Enter oxidized hyaluronic acid to be crosslinked.
3rd, the articular cartilage based on oxidized hyaluronic acid-II Collagen Type VIs and self concentration bone marrow nucleated cell repairs material
Expect the application in articular cartilage repair materials are prepared.
The beneficial effects of the present invention are：The present invention is disclosed based on oxidized hyaluronic acid-II Collagen Type VIs and self concentration bone
The articular cartilage repair materials of marrow karyocyte, the material is by oxidized hyaluronic acid, II collagen types and concentrates marrow self
Karyocyte is made, and the self concentration bone marrow nucleated cell of implantation has self-renewing and into cartilage differentiation potential, participates in cartilage
Defect repair, therefore material obtained has cartilage damage good repairing effect, the transparent cartilage sample of repair tissue is repaired
Tissue surface flatness, with the degree of integration of neighbouring normal cartilage, II collagen types content, GAG contents, calcified cartilage layer and
Subchondral bone form etc. is significantly better than that blank control group, there is the clinical potential converted.
Description of the drawings
In order to make the purpose of the present invention, technical solution and advantageous effect clearer, the present invention provides drawings described below and carries out
Explanation：
Fig. 1 is SDS-PAGE electrophoresis (Lane1, Lane2 and Lane3 represent different applied sample amounts respectively).
Fig. 2 shakes bacterium experiment for II collagen type hydrogels.
Fig. 3 is mesenchymal stem cell figure (A：Cultivate 2 days adherent mescenchymal stem cells；B：The mesenchyma of passage P3 is done
Cell).
Fig. 4 is sufficiently mixed figure (A for II Collagen Type VIs with mescenchymal stem cell：II Collagen Type VIs and cell co-culturing, inducing；B：It is solidifying
Glue is co-cultured with mescenchymal stem cell).
Fig. 5 is HA and OHA Fourier infrared spectrums.
Fig. 6 is CCK-8 testing results.
Fig. 7 is OHA-COLII-BMSC plastic results in a mold.
Fig. 8 induces result (A for three system of pig bone bone marrow-drived mesenchymal stem：Mescenchymal stem cell osteogenic induction；B：Mesenchyma is done
Cell adipogenic induction；C：Mescenchymal stem cell chondrocyte induction).
Fig. 9 into gel and cultivates different time section result (A for OHA-COLII-BMSC：OHA-COLII-BMSC is into solidifying
Cementing fruit；B：Cultivate 14 it is small when after cut into slices result；C：Cultivate 21 it is small when after cut into slices result；D：Gel slice result before culture；E：
Cultivate 14 it is small when gel slice result；F：Cultivate 21 days gel slice results).
Figure 10 is cartilage repair tissue appraisal result (A：MOCART appraisal results；B：O`Driscoll appraisal results；C：
ICRS appraisal results).
Figure 11 is the preparation of oxidized hyaluronic acid and generates cross-linking reaction schematic diagram with II collagen types.
Specific embodiment
Below in conjunction with attached drawing, the preferred embodiment of the present invention is described in detail.
The preparation of embodiment 1, the bionical gel of oxidized hyaluronic acid II Collagen Type VIs
(1) extraction and identification of cartilage typeⅡ Collagen hydrogel
A. gartilage powder is prepared：Fresh Guizhou minipigs knee joint hyaline cartilage is obtained, it, will be cold in -20 DEG C of refrigerator freezing 12h
Freeze cartilage to combine in glass dish, be placed in vacuum freeze drier, freeze (temperature is down to -66 DEG C, and pressure is down to 1Pa) afterwards for 24 hours,
Cartilage piece after freeze-drying pours into liquid nitrogen cryogenics pulverizer, is persistently filled with liquid nitrogen, and cartilage piece is ground into dry cartilage powder, receives
Collect gartilage powder to hermetic bag to preserve for use；
B. the extraction of typeⅡ Collagen：It weighs 20g and freezes gartilage powder and be placed in beaker, pour into 1L 4M guanidine hydrochloride solutions
Stirring is suspended, and magnetic stirring apparatus, which persistently stirs, to be placed in 4 DEG C of refrigerators, digestion gartilage powder for 24 hours after mucopolysaccharide in matrix to even
Pulpous state state；After digestion completely, digestion product is centrifuged into 8000rpm × 20min at 4 DEG C, abandons supernatant, collects sediment to beaker
In, it adds water at 1500ml scales, then measures 60ml acetic acid and add in beaker, add in 2g pepsins, add water to 2000ml
At scale, pH value is adjusted between 2.5-3, is placed in refrigerator magnetic agitation under the conditions of 4 DEG C and is digested 48h；Pepsin digestion is complete
Finish after centrifuging 8000rpm × 20min in vertical high speed centrifugal machine, abandon precipitation, collect supernatant；Then into supernatant slowly
Pour into 1.5M NaOH solutions parallel stirring to pH value be 7.5 when stop fall note, add in NaCl make its final concentration of 3M, 4 DEG C of conditions
Under, 8000rpm × 20min is centrifuged, collects sediment；Sediment is dispensed into bag filter (diameter 25mm, molecular weight 3500), and two
End is tightened with nylon wire, and distilled water is dialysed, and preceding 48h replaces a dialyzate per 6h, is replaced once per 12h afterwards, always during dialysis
Between about 5d.Conductivity meter monitors conductivity of dialysate in real time, treats conductivity of dialysate, less than physiological saline, close to pure water electricity
Terminate to dialyse during conductance；Sticky colloidal solution in bag filter is moved into concentrator bowl, under the conditions of 4 DEG C, the centrifugation of vertical high speed centrifugal machine
8000rpm × 20min collects sediment into sterile volumetric flask, adds in the hydrochloric acid solution of 0.15mol/L, white after dissolving 3 days
Protein precipitation disperses, dissolves in gelatinous, stirs evenly, and in super bacterium workbench, dispenses to different capabilities sterilizing glass capsulation
In bottle, 4 DEG C preserve for use；
Appropriate collagen hydrogels is taken to be freezed, by formula (weight in wet base-volatilised liq weight)/(weight in wet base volume), meter
II Collagen Type VI hydrogel w/v are calculated, the results show that II collagen types detection mass fraction is made using the method for the present embodiment
For 10% (w/v).
By the II collagen types of extraction after PAGE gel electrophoresis, the results are shown in Figure 1.The results show that it carries
The albumen taken only shows a band, and with increasing for applied sample amount, band concentration in gradient enhances, band molecular weight about 120KD,
For the peculiar protein band of cartilage II collagen types, without apparent miscellaneous band development on other molecular weight, illustrate the II types extracted
Collagen purity is higher.
It after II collagen types hydrogel adjusts neutrality with 2M NaOH, is placed in LB culture mediums, repeatedly carries out shaking bacterium reality
It tests, though there is part collagenolysis bottom in culture medium, shakes tube middle and upper part culture medium transparent clear, with positive and negative tube
Comparison, all shows pollution-free negative findings (Fig. 2).
After medulla mesenchyma xeromenia crosses density gradient centrifugation, separated cell culture medium is resuspended, and culture 2d changes liquid punching
Floating lymphocyte and red blood cell are washed off, adherent cell is the mesenchymal stem cell obtained, and cell is in polygonal, length
Fusiformis, primary cell cell division form multiple cell colonies as common (A in Fig. 3) after 4-5 days.After secondary culture,
Cell growth is more vigorous, and spindle shape cell can form 80%-90% cell fusion degree through 5-7 days in blake bottle, in volume
Knit shape, circinate arrangement.(B in Fig. 3)
After II Collagen Type VIs are sufficiently mixed with mescenchymal stem cell, gel piece can be immersed in culture medium, and a small amount of gel piece can
It dissolves, but it is simultaneously muddy caused by uncontamination, it need to carefully be replaced when replacing culture medium, avoid blowing and beating, gel piece is connected with physics
Reunion is connect, intensity is relatively low, loosely organized (A in Fig. 4).Visible arrangement of collagen fibers of taking a picture under an optical microscope is loose, solidifying
The visible silk sample collagenous fibres in blob of viscose edge, length is homogeneous, rounded in the cell of gel piece, comes off and becomes in the cell on culture plate
Extend, start division growth.(B in Fig. 4)
(2) oxidation of hyaluronic acid
The oxidation of hyaluronic acid：It takes in 4g hyaluronic acids dissolving 200ml pure water, it is completely molten that 6h is stirred on magnetic stirring apparatus
Solution is configured to the hyaluronic acid solution that mass fraction is 2%, for use；2g sodium metaperiodates is taken to add in 20ml pure water after dissolving, are fallen
Lasting to stir in 2% hyaluronic acid solution for entering preparation, aluminium-foil paper package is protected from light, complete oxygen under room temperature (18~25 DEG C) environment
When change reaction 6 is small；It measures ethylene glycol 20ml and adds in beaker and react 1h, terminate oxidation reaction, reaction solution is poured into bag filter
(diameter 25mm, molecular weight 3500), both ends tighten, and distilled water dialysis, preceding 48h replaces a dialyzate per 6h, afterwards per 12h more
It changes 1 time.Conductivity meter monitors conductivity of dialysate in real time, treats conductivity of dialysate, and close to pure water conductivity when terminates to dialyse, and obtains
Oxidized hyaluronic acid is obtained, dialyzate is poured into lyophilized plate and is placed in -20 DEG C of refrigerators, after precooling 12h freezes, it is cold to be placed in vacuum
(temperature is down to -66 DEG C, and pressure is down to 1Pa) for 24 hours is freeze-dried in lyophilizer, finally by dry oxidized hyaluronic acid with
100mg is dispensed for unit into EP pipes, sealant tape sealing, 60 irradiation sterilizations of Co.
Hyaluronic acid transparent liquid after sodium periodate oxidation, in spongy after freezing, by lyophilized hyaluronic acid and
Inoxidized hyaluronic acid, through pellet technique, the detection of row Fourier infrared spectrum occurs characteristic in wavelength 1750 and changes
Become, show that the hydroxyl on hyaluronic acid is oxidized to aldehyde radical (Fig. 5) by sodium metaperiodate specificity.
Hyaluronic acid after oxidation can be dissolved completely in culture medium, pig bone bone marrow-drived mesenchymal stem in 0.5mg/ml,
After culture 48h being carried out in 1.0mg/ml, 1.5mg/ml oxidized hyaluronic acid cultivating system, row CCK-8 detections, with blank control group
Carry out control no significant difference, it was demonstrated that it weakens (Fig. 6) without apparent cytotoxicity and proliferation activity.
(3) preparation of the bionical gel of oxidized hyaluronic acid II Collagen Type VIs
A. the oxidized hyaluronic acid 100mg of irradiation sterilization is taken, 1ml PBS solutions is added in, it is made to be completely dissolved for use.
B. 1g II collagen types are taken, add in 75 μ l NaOH (2M), nerve dissector stirs evenly, and liquid-transfering gun adds in
100 μ l dissolve oxidized hyaluronic acid, are sufficiently stirred.It is filled with into stainless steel mould hole (diameter 8mm, thick 3mm), after 2min
Oxidized hyaluronic acid II collagen types (OHA-COLII) plastic completely is in solidification shape.After being placed in -20 DEG C of refrigerators pre-freeze for 24 hours,
It is placed in vacuum freeze-drying machine and carries out lyophilized being in spongy.It send to the 3rd central laboratory of medical university, ion sputtering instrument is to sample
Plate 10nm golden films, scanning electron microscopic observation section.Remaining gel piece is dipped in the culture dish of physiological saline, is placed in 37 DEG C of incubators
Cultivate 14d, observation swelling and dissolving situation (Fig. 7).
Three system of pig bone bone marrow-drived mesenchymal stem induces：Take the mescenchymal stem cell of secondary culture.
A) osteogenic induction：When cell fusion degree reaches 80-90%, digested with the pancreatin of mass fraction 0.25%；
By the stem cell digested according to 2 × 104cells/cm2Cell density be inoculated with six orifice plates in, inoculation before will be put into orifice plate
The coated slide of 0.1% gelatin adds in 2mL complete mediums per hole.It, will be in hole completely when cell fusion degree reaches 60-70%
Culture medium siphons away.2mL human bone marrow mesenchymal stem cells Osteoinductive differentiation complete mediums are added in into six orifice plates respectively, every
3d changes liquid, and 3w hystazarins are red is dyed for induction.As a result as shown in A in Fig. 8, the results show mesenchymal stem cell pass through into
After self-bone grafting, the calcium tubercle of secretion is by the red dye of alizarin.
B) adipogenic induction：Cell inoculation method is same as above, and cell fusion degree reaches 100%, and interstital stem cell is cultivated completely
Base siphons away, and 2ml mesenchymal stem cell adipogenic induction differential medium A liquid is added in into six orifice plates.After inducing 3d, six are siphoned away
A liquid in orifice plate adds in 2ml bone marrow interstital stem cell adipogenic induction differential medium B liquid.After for 24 hours, B liquid is siphoned away, gains A liquid
It is induced.After A liquid and B liquid alternating action 4 times (15d), continue with B liquid culture 5d to be maintained to become sufficiently large, circle until fat drips,
Oil red O stain after fixation.As a result as shown in B in Fig. 8, after adipogenic induction, cell body becomes larger the results show, cell paving
Exhibition, the light dye of nucleus, cytoplasm is interior to generate substantial amounts of lipochondrion, and larger lipochondrion can merge, and form fat drop.
C) into chondrocyte induction：Cell inoculation method is same as above, and cell fusion degree reaches 100%, and interstital stem cell is trained completely
Foster base siphons away, and 2ml mesenchymal stem cells are added in into six orifice plates into chondrocyte induction differential medium, 21d are cultivated, after fixed
The blue dyeing of aricine.Slide in six orifice plates is taken out with tweezers after dyeing, observation induction situation of taking pictures under an optical microscope；As a result
As shown in C in Fig. 8, into after chondrocyte induction, mescenchymal stem cell form changes the results show, becomes paving stone from spindle shape
Sample, secretion GAG is dyed light blue by quinovatine orchid, and nucleus is not colored.
Culture is taken to the pig bone bone marrow-drived mesenchymal stem of the third generation, outwells culture medium, after PBS is rinsed 2 times, 0.25% pancreatin
3min is digested, cell is in spherical, pats the suspension of bottle wall cell detachment, and 2ml complete mediums terminate digestion.1000rpm×3min
After centrifugation, 2ml culture mediums are resuspended, and cell counting count board counts cell, takes 5 × 106It is centrifuged again into 2ml EP pipes.1g is taken to be adjusted to
Neutral COLII is added in the EP pipes of centrifuge cell, is added in 100 μ l dissolving OHA, is stirred, will be gel-filled supreme
In stainless steel mould after temperature disinfection, its gelled, i.e. OHA-COLII-BMSC are treated after 2min.It is placed in complete medium,
It is cultivated in the orifice plate.The result shows that OHA-COLII-BMSC can be in sterile mold well into gel (A in Fig. 9).
It is cultivated in the orifice plate to 14d, after 21d takes sample, 10% formalin to fix for 24 hours respectively, row HE dyeing, in optics
Micro- Microscopic observation section situation；Cultivating to 14d takes one piece of glutaraldehyde of sample to fix, 50%, 70%, 80%, 90% ethyl alcohol
Each 15min of serial dehydration, then with 100% ethanol dehydration 3 times, each 30min；The tert-butyl alcohol replaces each 30min, replaces 3 times altogether.
Freeze drier drying sample is adhered to sample on sample stage with double faced adhesive tape, and ion sputtering instrument plates 10nm golden films to sample, sweeps
Retouch electron microscopic observation sample section situation.The results are shown in Figure 9, the results show that still being maintained in the medium after culture 14d, 21d
Complete (B and C in Fig. 9) of gel, section show that mesenchymal cell is uniformly distributed in gel piece, without significantly reduce (D in Fig. 9,
E and F).It is dehydrated after fixation, gel piece pyknosis is in lumps, and section surface sweeping Electronic Speculum shows that pyknosis sphaerocyst is distributed in gel
(A in Fig. 9).
The compound self concentration bone marrow nucleated cell of embodiment 2, bionical gel repairs cartilage defect experiment
In order to explore the effect of the compound self concentration bone marrow nucleated cell repairing articular cartilage damage of bionical gel, devise
One-step method repairs the zoopery of cartilage defect, and pig knee joint is repaired by the compound self concentration bone marrow nucleated cell of bionical gel
Cartilage defect.Using Guizhou minipigs as experimental animal, suction obtains marrow at ilium ridge first under general anesthesia, is separated by marrow
Liquid separation obtains self concentration bone marrow nucleated cell, and is mixed with II collagen types, adds oxidized hyaluronic acid and is handed over
Connection.The complete thick cartilage defect model of a diameter of 8mm is prepared at knee joint femoral coaster by modus operandi, by cell mixing
Hydrogel is implanted into cartilage defect.Postoperative 1,3, June seen by MRI, gross examination of skeletal muscle, stereomicroscope and pathological section
Examine analysis.By this part zoopery, the experiment effect that the strategy repairs cartilage defect is explored, verifies the feasible of the strategy
Property, provide basis for the clinical application in later stage.
Specific experiment method is as follows：
40 Guizhou minipigs are randomly divided into two groups of A, B according to table of random numbers method, every group of 20 knees, totally 40 knee, avoids repairing
Compound articulation secondary lesion, only a lateral joint are performed a surgical operation；A groups are blank control group (simple hyaline cartilage defect group)；B groups are
Experimental group (the compound autologous bone marrow concentration karyocyte reparation group of II collagen types-oxidized hyaluronic acid hydrogel)；Every group of knee
Joint is observed, detected and is evaluated in 4 time points (1,3,6 and December) materials, and each time point of drawing materials chooses 5 knees passes
Save sample.
Guizhou minipigs concentrate the acquisition of bone marrow nucleated cell self：Sleep peaceful 4ml of intramuscular injection land carries out basal anaesthesia, and 3% penta
Barbital sodium solution is injected by 0.2mL/kg dosage ear vein, anaesthetizes Guizhou minipigs；Postanesthetic Guizhou minipigs are placed in
On operating table, lateral position, offside anterior superior spine area preserved skin, disinfection, drape are taken.It is pierced using No. 16 marrow puncture needles at crista iliaca
Enter, feel that inserting needle about 1cm, taking-up needle core are injected with the 20ml that heparin (1000U/ml) has been used to prewet again after the sense that falls through
Device (heparin 2ml) bone marrow extraction blood.According to the position of how much appropriate adjustment puncture needles of aspiration and depth, aspirate altogether about
20ml send laboratory to carry out self concentration bone marrow nucleated cell separation；The anti-freezing Guizhou minipigs bone marrow fluid of extraction is injected
In 50ml centrifuge tubes, isometric sterile PBS liquid, mixing are added.Bone marrow fluid after mixing is slowly injected into along centrifugation tube wall
To in containing isometric Percoll (1.073g/ml) cell separating liquid, injection process slowly carries out, and keeps separating liquid with mixing
Interface is complete between liquid.It prevents from because of excessive velocities bone marrow fluid being made to mix with Percoll cell separating liquids, influences separation effect
Fruit.Self concentration bone marrow nucleated cell is obtained using Percoll density-gradient centrifugation methods：Mixing is centrifuged with the speed of 2000rpm
Liquid 15min, it is seen that liquid layered phenomenon：It is respectively from top to bottom：Plasma layer, karyocyte layer (are done carefully containing medulla mesenchyma
Born of the same parents), transparent separation liquid layer and red blood cell layer.Careful karyocyte layer (the 2nd layer of white cloud layer) of drawing is to new centrifugation
Pipe adds in the karyocyte that the washing of 10ml PBS solutions is drawn, with the speed centrifuge washing cell 5min of 1500rpm, abandons supernatant.
10ml PBS piping and druming washing cells are added in again, and 1000rpm speed centrifugation 5min abandons supernatant.It adds in 5ml PBS to be resuspended, and makes
With cell counting count board row cell count, it is spare to collect cell；The compound marrow concentration karyocyte of II collagen types：Take 2 × 106
A bone marrow nucleated cell, is placed in EP pipes, and the speed centrifugation 5min of 1000rpm abandons supernatant, adds in 100mg II collagen types and mix
It closes uniformly, is shaken and centrifuged by whirlpool, it is for use to remove bubble in mixed gel.
Then animal surgery is carried out, is as follows：
A. bone marrow extraction is after offside knee hair cutter cropping preserved skin, and preserved skin range size is about 40cm × 30cm, with nothing
Bacterium gauze dips in anti-bacterial hand lotion, and to cleaning without dirt, Iodophor pot sprays art area in ecouvillonnage area repeatedly.Operated cohort is placed in animal
On the V-shaped groove of operating table, lateral position is taken, Iodophor spray disinfectant again spreads disposable sterilized single towel.It takes by kneecap to enter on the outside of knee joint
Road, longitudinal incision skin are about 6cm, successively cut subcutaneous tissue, capsular ligament, by the inside lateral dislocation of kneecap, appear femoral bone pulley；
B. with a diameter of 8mm self-control cartilage defect sleeve in femoral bone pulley bore an annular cartilage defect, depth with
Articular hyaline cartilage layer thickness is consistent.Hyaline cartilage in ring is shredded in netted using eye scissors, small curet strike off shred it is saturating
Bright cartilage, and ensure the complete of calcified cartilage layer structure, and ensure that substrate is oozed out without marrow blood, modify the bottom and side of defect
Edge prepares the complete thick cartilage defect model of a diameter of 8mm；
C. rinsing modeling region with physiology salt Aquapulse gun, cartilage is broken to be cut to remove, and 10 μ l oxidized hyaluronic acid solution are added
In the II collagen type gels for entering compound cells.Nerve dissector is implanted into cartilage defect after stirring evenly, and moulding makes
Gel surface is concordant with cartilage surface, strikes off extra gel around, stands 2min and treats the abundant plastic of gel composite.Reset kneecap
And knee joint is shown in gel piece adhesion-tight to passive activity for several times, resets kneecap absorbable thread and strengthens suture inside support band afterpulse
It rinses, layer-by-layer suture is subcutaneous and skin；
D. immediate postoperative gives deer peaceful 1 intramuscular injection of waking up to waken, 4,000,000 unit intramuscular injection of benzylpenicillin potassium prevention infection for animals.
Postoperative knee joint does not fix, daily art area iodophor disinfection 2 times, intramuscular injection antibiotic 2 times (4,000,000 units of benzylpenicillin potassium for animals).
Regular diet is raised, daily to observe animal basic condition, wound healing situation and walking step state etc..Postoperative 4 Guizhou minipigs
Depart from research series due to anaesthetizing associated death (n=2), abarticulation (n=1) and postoperative infection (n=1).Therefore, altogether
36 Guizhou minipigs complete whole operations and clinical follow assessment, therefore this research temporarily terminates postoperative 12 months
Long term follow-up, only the follow-up repairing effect of postoperative 1,3 and June.Remaining 36 Mini-musk swines return to normal for 7 days after surgery
Walking step state, postoperative 10 days or so operative incisions reach Wound healing by first intention.Follow-up in January group, materials time are postoperative 33 ± 3 days；March
Follow-up group, materials time are postoperative 92 ± 1 days；Follow-up in June group, materials time are postoperative 183 ± 5 days.
(1) MRI scores
Using improvement cartilage repair tissue MR observation points-scoring systems (Modified magnetic resonance
Observation of cartilage repair tissue score, MOCART), the repairing effect of cartilage defect is carried out
Sxemiquantitative is scored, and specific standards of grading are as shown in table 1：
Table 1. improves cartilage repair tissue MR observation points-scoring systems (MOCART)
Its appraisal result is as shown in A in table 2 and Figure 10, the results show that blank control group and experimental group after surgery 1,3 and 6
The scoring of moon MOCART is respectively 10 ± 0 and 53.5 ± 4.74 (n=10, p<0.001)；42 ± 4.83 and 89.5 ± 5.99 (n=
10,p<0.001)；And 64 ± 6.99 and 96 ± 5.16 (n=10, p<0.001).
2. cartilage repair tissue MOCART of table scores
Value is represented as average value ± SD, Mann-Whitney ' s U-test was used.*p<0.05
(Treat.Vs.Cont.)
aIn original MOCART, subcartilaginous osseous lamella substitutes by calcified layer
The result shows that (the II collagen types-compound the autologous bone marrow of oxidized hyaluronic acid hydrogel prepared using the present invention
It is more preferable to concentrate karyocyte reparation cartilage effect.
(2) O ' Driscoll histological scores are improved：Using improvement O ' Driscoll histological scores to 2 groups of experimental animals
Cartilage defect repair effect carries out semi-quantitative analysis, as a result as shown in B in table 3 and Figure 10.Control group and treatment group after surgery 1,3
And improvement in June O ' Driscoll histological scores are respectively：0 vs 13.3 ± 2.21 (n=10, p<0.001)；6.1±2.02
Vs 16.8 ± 1.4 (n=10, p<And 8 ± 1.49 vs 17.6 ± 1.26 (n=10, p 0.001)<0.001).Surface treatment group
Damaged cartilage repairing effect is more excellent compared with control group.
3. cartilage repair tissue of table improves O ' Driscoll histological scores
Value is represented as average value ± SD, Mann-Whitney ' s U-test was used.*p<0.05
(Treat.Vs.Cont.)
(4) ICRS histological scores are improved：Cartilage defect repair effect is commented using improvement ICRS histological scores
Valency is analyzed, from defect repair degree, cambium and neighbouring cartilage integration, substantially repair tissue color, sight and repair tissue
Mechanical strength etc. carries out analysis and assessment, and the results are shown in Table 4.Control group and treatment group 1,3 and improvement in June ICRS after surgery
Histological score is respectively：0 vs 16.6 ± 1.78 (n=10, p<0.001)；11.3 ± 2.71 vs, 19.3 ± 1.64 (n=
10,p<And 15.1 ± 1.85 vs 19.9 ± 0.32 (n=10, p 0.001)<0.001).
4. cartilage repair tissue of table improves ICRS histological scores
Values were presented as mean±SD,Mann-Whitney’s U-test was used.*p<
0.05(Treat.Vs.Cont.)
The above results show have the core thin by the compound self concentration marrow of the bionical gel of oxidized hyaluronic acid-II Collagen Type VIs
Born of the same parents have good repairing effect to Guizhou minipigs cartilage defect.Cartilage defect region is filled out by cartilage sample new life repair tissue
It fills, repair tissue surfacing, is integrated with neighbouring normal cartilage good.Cell is similar to normal chondrocyte form in matrix,
It can be seen that separating phenomenon, there is self-renewal capacity, mescenchymal stem cell is into cartilage differentiation.II Collagen Type VIs, GAG contain in repair tissue
For amount apparently higher than blank control group, MOCART scorings, O ' Driscoll histological scores and ICRS histological scores are also apparent
Better than blank control group, there is the clinical potential converted.Its principle is：Hyaluronic acid is modified hyalomitome by sodium periodate oxidation
Its hydroxyl is oxidized to aldehyde radical by acid, and the aldehyde radical in oxidized hyaluronic acid passes through Schiff's with the amino on II collagen types
Base reacts, and chemical crosslinking occurs into gel (shown in Figure 11), the method overcome hyaluronic acid cannot be with II Collagen Type VI eggs
White hair gives birth to the problem of polymerisation.
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (8)
1. the articular cartilage repair materials based on oxidized hyaluronic acid-II Collagen Type VIs and self concentration bone marrow nucleated cell, special
Sign is, is made of oxidized hyaluronic acid, II collagen types and self concentration bone marrow nucleated cell.
2. the joint based on oxidized hyaluronic acid-II Collagen Type VIs and self concentration bone marrow nucleated cell according to claim 1
Cartilage repair material, it is characterised in that：The addition of oxidized hyaluronic acid, II collagen types and self concentration bone marrow nucleated cell
Amount is by every 2 × 106A self concentration bone marrow nucleated cell adds in 100mg II collagen types gels and 10 μ l concentration are
The oxidized hyaluronic acid solution of 100mg/ml.
3. according to articular cartilage of the claim 1 based on oxidized hyaluronic acid-II Collagen Type VIs and self concentration bone marrow nucleated cell
Repair materials, it is characterised in that：The oxidized hyaluronic acid of the 100mg/ml adds in 1ml by every 100mg oxidized hyaluronic acid
PBS solution, it is obtained through being completely dissolved.
4. there is the core thin based on oxidized hyaluronic acid-II Collagen Type VIs and self concentration marrow according to any one of claims 1 to 3
The articular cartilage repair materials of born of the same parents, it is characterised in that：The II collagen types are prepared by following methods：Lyophilized gartilage powder is pressed
Mass volume ratio is 20:1(g：L it is) that the stirring of 4M guanidine hydrochloride solutions is suspended with concentration, stirring is placed under the conditions of 4 DEG C, and digestion is soft
Mucopolysaccharide in bone meal matrix is to being homogenized state；After digestion completely, digestion product is centrifuged under the conditions of 4 DEG C, 8000rpm
20min abandons supernatant, collects sediment, is dissolved in water, and then adds in the acetic acid for being equivalent to 3/50 times of guanidine hydrochloride solution volume,
The pepsin for being equivalent to lyophilized 0.1 times of gartilage powder weight is added in, final volume is added water to as 2 times of guanidine hydrochloride solution, adjusts pH
It is worth between 2.5-3, then digests 48h under the conditions of 4 DEG C；Digestion is finished to be centrifuged after 8000rpm in vertical high speed centrifugal machine
20min abandons precipitation, collects supernatant；Then the NaOH solution that concentration is 1.5M is poured slowly into supernatant and is stirred to pH
Be worth for 7.5 when stop falling to note, adding in NaCl makes its final concentration of 3M, and under the conditions of 4 DEG C, 8000rpm centrifugation 20min collect precipitation
Object；Sediment, which is dispensed, treats conductivity of dialysate into bag filter, and close to pure water conductivity when terminates to dialyse；It will be sticky in bag filter
For colloidal solution under the conditions of 4 DEG C, vertical high speed centrifugal machine 8000rpm centrifugation 20min collect sediment into sterile volumetric flask,
The hydrochloric acid solution of 0.15mol/L is added in, white precipitate albumen disperses, dissolves in gelatinous after dissolving 3 days, stirs evenly, and dispenses,
It is lyophilized, it is spare.
5. there is the core thin based on oxidized hyaluronic acid-II Collagen Type VIs and self concentration marrow according to any one of claims 1 to 3
The articular cartilage repair materials of born of the same parents, which is characterized in that the oxidized hyaluronic acid is prepared by following methods：It is configured to mass fraction
For 2% hyaluronic acid solution, the sodium periodate solution of 0.1g/ml is then added in, is stirred, complete oxidation under the conditions of being protected from light
6 it is small when, then add in ethylene glycol and terminate reaction, then when reaction solution is poured into bag filter to electrical conductivity close to pure water terminates
Analysis obtains oxidized hyaluronic acid.
6. there is the core thin based on oxidized hyaluronic acid-II Collagen Type VIs and self concentration marrow according to any one of claims 1 to 3
The articular cartilage repair materials of born of the same parents, which is characterized in that self concentration bone marrow nucleated cell is prepared by following methods：To current animal
It is anaesthetized, then with the syringe bone marrow extraction blood prewetted using 1000U/ml heparin, then by the anti-freezing bone of extraction
Marrow liquid adds in isometric sterile PBS liquid, mixing, bone marrow fluid is slowly injected into along tube wall after mixing is containing isometric concentration
In Percoll born of the same parents' separating liquid of 1.073g/ml, interface is complete between injection process holding separating liquid and mixing liquid, then
Self concentration bone marrow nucleated cell is obtained using Percoll density-gradient centrifugation methods, mixed liquor is first centrifuged with the speed of 2000rpm
Body 15min draws karyocyte layer, the karyocyte that PBS solution washing is drawn is added in, with the speed centrifuge washing of 1500rpm
Cell 5min, abandons supernatant；PBS piping and druming washing cells are added in again, and 1000rpm speed centrifugation 5min abandons supernatant, adds in PBS weights
It hangs to obtain the final product.
7. bone marrow nucleated cell is concentrated described in any one of claim 1~6 based on oxidized hyaluronic acid-II Collagen Type VIs and self
The preparation method of articular cartilage repair materials, which is characterized in that be as follows：To concentrate self bone marrow nucleated cell first with
II collagen types mix, and then add oxidized hyaluronic acid and are crosslinked.
8. application of any one of the claim 1~6 articular cartilage repair materials in articular cartilage repair materials are prepared.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711446501.2A CN108096632B (en) | 2017-12-27 | 2017-12-27 | Articular cartilage repair materials and preparation method based on oxidized hyaluronic acid-II Collagen Type VI and self concentration bone marrow nucleated cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711446501.2A CN108096632B (en) | 2017-12-27 | 2017-12-27 | Articular cartilage repair materials and preparation method based on oxidized hyaluronic acid-II Collagen Type VI and self concentration bone marrow nucleated cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108096632A true CN108096632A (en) | 2018-06-01 |
| CN108096632B CN108096632B (en) | 2019-02-05 |
Family
ID=62213872
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711446501.2A Active CN108096632B (en) | 2017-12-27 | 2017-12-27 | Articular cartilage repair materials and preparation method based on oxidized hyaluronic acid-II Collagen Type VI and self concentration bone marrow nucleated cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108096632B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109589451A (en) * | 2018-11-19 | 2019-04-09 | 浙江星月生物科技股份有限公司 | A kind of one-step method regenerating bone or cartilage repairing composite and preparation method thereof |
| CN113476657A (en) * | 2021-06-21 | 2021-10-08 | 四川大学 | Collagen-based hydrogel for cartilage tissue engineering and preparation method thereof |
| CN114870092A (en) * | 2022-05-20 | 2022-08-09 | 广州骊贝生物科技有限公司 | Bone substitute complex and preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6623963B1 (en) * | 1999-12-20 | 2003-09-23 | Verigen Ag | Cellular matrix |
| CN1539514A (en) * | 2003-11-03 | 2004-10-27 | 刘永庆 | Method for preparing multifunctional biological repair material |
| CN101091804A (en) * | 2007-07-18 | 2007-12-26 | 戴江华 | Method for constructing tissue-engineering bone by pouring stem cell of inducing bone marrow ground substance into vivo |
| CN101301490A (en) * | 2008-06-27 | 2008-11-12 | 四川大学 | Method for preparing medical level type II collagen |
| CN102188748A (en) * | 2011-04-29 | 2011-09-21 | 成都军区昆明总医院 | Novel chondrocyte epimatrix membrane and preparation method thereof |
| CN102488925A (en) * | 2011-12-29 | 2012-06-13 | 成都普川生物医用材料股份有限公司 | Injectable articular cartilage tissue repair material and its preparation method |
-
2017
- 2017-12-27 CN CN201711446501.2A patent/CN108096632B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6623963B1 (en) * | 1999-12-20 | 2003-09-23 | Verigen Ag | Cellular matrix |
| CN1539514A (en) * | 2003-11-03 | 2004-10-27 | 刘永庆 | Method for preparing multifunctional biological repair material |
| CN101091804A (en) * | 2007-07-18 | 2007-12-26 | 戴江华 | Method for constructing tissue-engineering bone by pouring stem cell of inducing bone marrow ground substance into vivo |
| CN101301490A (en) * | 2008-06-27 | 2008-11-12 | 四川大学 | Method for preparing medical level type II collagen |
| CN102188748A (en) * | 2011-04-29 | 2011-09-21 | 成都军区昆明总医院 | Novel chondrocyte epimatrix membrane and preparation method thereof |
| CN102488925A (en) * | 2011-12-29 | 2012-06-13 | 成都普川生物医用材料股份有限公司 | Injectable articular cartilage tissue repair material and its preparation method |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109589451A (en) * | 2018-11-19 | 2019-04-09 | 浙江星月生物科技股份有限公司 | A kind of one-step method regenerating bone or cartilage repairing composite and preparation method thereof |
| CN109589451B (en) * | 2018-11-19 | 2022-03-08 | 浙江星月生物科技股份有限公司 | One-step cartilage regeneration and repair compound and preparation method thereof |
| CN113476657A (en) * | 2021-06-21 | 2021-10-08 | 四川大学 | Collagen-based hydrogel for cartilage tissue engineering and preparation method thereof |
| CN114870092A (en) * | 2022-05-20 | 2022-08-09 | 广州骊贝生物科技有限公司 | Bone substitute complex and preparation method and application thereof |
| CN114870092B (en) * | 2022-05-20 | 2023-08-22 | 广州骊贝生物科技有限公司 | Bone substitute complex, preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108096632B (en) | 2019-02-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107281550B (en) | Preparation method of co-crosslinked double-network hydrogel scaffold for promoting cartilage injury repair | |
| Har-Shai et al. | An integrated approach for increasing the survival of autologous fat grafts in the treatment of contour defects | |
| CN103079577B (en) | The preparation technology of wound restoration agent compositions, pipe and device | |
| CN110975010B (en) | Placenta tissue matrix material and preparation method thereof | |
| CN106456837A (en) | Methods for preparation of a terminally sterilized hydrogel derived from extracellular matrix | |
| CN109054047A (en) | A kind of silk gum/graphene oxide composite hydrogel and its preparation method and application | |
| CN108096632B (en) | Articular cartilage repair materials and preparation method based on oxidized hyaluronic acid-II Collagen Type VI and self concentration bone marrow nucleated cell | |
| CN110496241A (en) | It is derived from the biomaterial and its preparation method and application of adipose tissue | |
| CN107735113A (en) | Extracellular matrix and its injectable formulation derived from cardiac fibroblast for treating ischemic disease or damage | |
| CN104056304B (en) | The DBM support repairing articular cartilage material of growth factor-loaded chitosan microball | |
| CN108057116A (en) | Application of the stem cell composition in skin injury medicine | |
| CN108057014A (en) | A kind of preparation method of the stem cell medicine of beauty and skin care | |
| BR112020015616A2 (en) | COMPOSITION OF BIOTINTA FOR DERME REGENERATION SHEET, METHOD FOR MANUFACTURING CUSTOMIZED DERME REGENERATION SHEET USING THE SAME AND CUSTOMIZED DERME REGENERATION SHEET MANUFACTURED USING THE MANUFACTURING METHOD | |
| CN106421917A (en) | Method for preparing composition for repairing cartilage injuries | |
| CN110522946A (en) | A kind of bone renovating material microballoon and preparation method thereof carrying rhBMP-2 | |
| CN107260639A (en) | A kind of gel rich in autologous growth factor repairs facial mask and preparation method thereof | |
| CN106729957A (en) | A kind of preparation method of the internal bleeding-stopping dressing with transglutaminase as crosslinking agent | |
| CN104622709B (en) | Human stem cell factor skin repair liquid and preparation method thereof | |
| CN110124107B (en) | PLGA cell scaffold for articular cartilage repair and preparation method and application thereof | |
| CN106474156A (en) | Purposes in preparing medicine for the compositionss | |
| CN108066750A (en) | Stem cell and its secretion are used to treat the new application of skin burn | |
| CN109182249A (en) | A kind of preparation method of the timbering material in body reparation cell transplantation | |
| CN108079363A (en) | A kind of kit and its application that cell processing is taken off for animal tissue | |
| CN114410583B (en) | Nerve/blood vessel network based on animal adipose tissue-derived hydrogel, and construction method and application thereof | |
| CN108355167A (en) | A kind of chitosan coating BCBB bone renovating bracket materials and preparation method thereof being sustained SDF-1 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |