CN106075584B - Syringeability cartilage takes off extracellular matrix mixing decalcified bone matrix hydrogel and preparation method thereof - Google Patents

Syringeability cartilage takes off extracellular matrix mixing decalcified bone matrix hydrogel and preparation method thereof Download PDF

Info

Publication number
CN106075584B
CN106075584B CN201610710756.4A CN201610710756A CN106075584B CN 106075584 B CN106075584 B CN 106075584B CN 201610710756 A CN201610710756 A CN 201610710756A CN 106075584 B CN106075584 B CN 106075584B
Authority
CN
China
Prior art keywords
cartilage
hydrogel
syringeability
mixing
extracellular matrix
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.)
Active
Application number
CN201610710756.4A
Other languages
Chinese (zh)
Other versions
CN106075584A (en
Inventor
黄洪超
马信龙
杨强
赵艳红
杨春梅
王连永
苗军
胡永成
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
TIANJIN HOSPITAL TIANJIN CITY
Original Assignee
TIANJIN HOSPITAL TIANJIN CITY
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by TIANJIN HOSPITAL TIANJIN CITY filed Critical TIANJIN HOSPITAL TIANJIN CITY
Priority to CN201610710756.4A priority Critical patent/CN106075584B/en
Publication of CN106075584A publication Critical patent/CN106075584A/en
Application granted granted Critical
Publication of CN106075584B publication Critical patent/CN106075584B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • A61L27/36—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]
    • 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
    • A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
    • 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
    • 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
    • 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
    • A61L27/52—Hydrogels or hydrocolloids
    • 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
    • A61L27/56—Porous materials, e.g. foams or sponges
    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • 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 syringeability cartilages to take off extracellular matrix mixing decalcified bone matrix hydrogel and preparation method thereof.Of the same race or xenogenesis cartilage is prepared cartilage cell epimatrix by de- cell processing by the present invention, of the same race or xenogenesis bone tissue is prepared into decalcified bone matrix by degreasing, decalcification, de- cell processing simultaneously, the two mix according to a certain percentage after using pepsin digestion, be solidified into syringeability hydrogel under neutral pH, 37 DEG C and suitable salt concentration conditions.Taking off extracellular matrix mixing decalcified bone matrix hydrogel using cartilage prepared by the method for the invention has good biodegradability, it is most important that has syringeability；Simultaneously as carrying out de- cell processing, immunogenicity is substantially reduced, and has preferable biocompatibility, and biocompatible reaction reduces；Present invention materials are simple, easy to operate, can be used for various cartilage defect fillings and the treatment of minimally invasive orthopaedics repair of cartilage, have good potential applicability in clinical practice.

Description

Syringeability cartilage takes off extracellular matrix mixing decalcified bone matrix hydrogel and its preparation Method
Technical field
The present invention relates to bioengineered tissue technology, specifically a kind of syringeability cartilage takes off extracellular matrix mixing decalcification Bone matrix hydrogel and preparation method thereof.
Background technique
Clinically articular cartilage damage, defect is caused to be always orthopaedics by factors such as regression, wound, various inflammation, tumours Clinical problems faced, articular cartilage overwhelming majority category hyaline cartilage, no blood supply, lymphoid tissue and innervation, self-regeneration Ability is limited, and self-regeneration ability is poor.Articular cartilage damage treatment becomes one of current Bones and joints surgery problem.
Clinically there are millions of patients to receive treatment because of articular cartilage damage every year.Native cartilage transplanting at present and allosome Cartilage transplantation all has certain limitation, and native cartilage transplanting generates new cartilage defect, and homogenous cartilage transplanting exists immune Originality reaction.Since the twentieth century eighties, cartilage cell treats articular cartilage damage and causes people's interest, i.e., soft Cartilage cell is transplanted at bone defect to realize the reparation of cartilage defect.But transplant cartilage cell merely, cell can not plant and Proliferation, causes ineffective.So for a long time, the regeneration of osteocartilaginous and subchondralo bone injury and the problem for repairing always puzzlement Orthopedic Clinical One of, and clinical and basic research one of hot spot.In recent years, with macromolecule medical material, biochemistry, materialogy, The multi-disciplinary fast development of molecular biology, preclinical medicine, clinical medicine, it is cartilage tissue engineered to be also rapidly developed, New approach is provided to solve this problem.
For these reasons, people are finding a kind of ideal cartilage repair material --- and antigenicity is low and lures with cartilage It leads and cartilage characteristic.The generally acknowledged reparation cartilage technology of home and overseas is that (Autologous Chondrocyte moves second generation ACI technology at present Plant): MACI technology (self cartilage compound cells epimatrix transplanting) has been used to clinic, specific method is exactly can be internal at present Then the compound cartilage cell of the timbering material of degradation is implanted to cartilage defect.
The late 20th century starts the research of de- cell tissue extracellular matrix, and cell is considered as the root of immunogenicity, The cell for sloughing tissue just removes most of immunizing antigen ingredient of tissue.Tissue can remove cell by de- cell step Film, cytoplasmic components and intracellular soluble protein etc. can induce the substance of immunological rejection, remaining extracellular matrix composition It has certain bioactivity.The acellular matrix much organized at present is as the timbering material in organizational project, such as cardiac muscle Extracellular matrix has injected treatment myocardial infarction and has been applied to clinic.
Minimally invasiveization trend is presented in surgical clinical surgical procedure.Bone surgery is also in this way, especially minimally invasive joint surgery development Rapidly, conventional cutting operation is avoided, the minimally invasive prosthetic of cartilage damage is more and more for syringeability bone-grafting material demand.
Hydrogel is the polymer with hydrophilic tridimensional network, can not be dissolved in water, have mobility and Good biocompatibility, has syringeability, and gelatinizing-in-situ is easy to operate.
Osteochondral tissue is made of cartilage cell and extracellular matrix, and extracellular matrix is more by II Collagen Type VI and aminoglucose Glycan (GAG) composition.About 70% moisture of articular cartilage, 20% II Collagen Type VI, 5% proteoglycan and 5% cartilage cell.Cartilage is thin Born of the same parents are located at the small chamber in cartilage matrix --- in cartilage cavities.Cartilage capsule is located at around lacuna, is rich in chondroitin sulfate.Cartilage is thin Born of the same parents are located in extracellular matrix by Derived from Mesenchymal Stem Cells.Articular chondrocytes epimatrix main component is proteoglycan, right Cell and its form have support and adjustment effect, adjust the signal transduction of the molecular levels such as cell metabolism, differentiation and proliferation.Joint Collagen accounts for the 90%~95% of cartilage collagen total amount based on typeⅡ Collagen in cartilage.
Bone tissue is hard connective tissue, is made of cell and matrix.Matrix is divided into organic matter and inanimate matter, organic matter Containing a large amount of collagenous fibres, inanimate matter is a large amount of inorganic calcium salt, and collagen is embedded in calcium salt matrix.
Collagen is the major structural protein of human body and vertebrate, is supporting tissue and connective tissue (skin, tendon forceps and bone The organic moiety of bone) main component, tropocollagen molecule structure be three polypeptide chains, be in three-dimensional spiral structure, have immunologic inertia, It is suitable as cartilage tissue engineering rack, can promote different type cell bonding, growth, differentiation and movement.Collagen is a kind of day Right hydrogel can meet the clinical needs to injectivity bone-grafting material.
Summary of the invention
The present invention is just to provide for a kind of new injectivity bone-grafting material to meet clinical needs, and the one kind proposed can Injectivity cartilage takes off extracellular matrix mixing decalcified bone matrix (DBM) hydrogel and preparation method thereof.
The present invention is realized according to following technical scheme.
A kind of syringeability cartilage takes off extracellular matrix mixing decalcified bone matrix hydrogel, the hydrogel be by of the same race or Xenogenesis cartilage is by de- cell processing, and of the same race or xenogenesis bone tissue is by degreasing, decalcification, de- cell processing, and the two is according to certain It is formed by after ratio mixing and is dissolved under weak acid, in physiological temp, neutral pH and suitable salinity (0.O1~0.1mol/ L being solidified under conditions of) has certain viscosity and the ossein hydrogel containing the osteogenic activity factor.
Described of the same race or xenogenesis cartilage and bone tissue are respectively derived from pig or ox or sheep or the cartilage and cancellous bone of people.
The hydrogel contains cartilage cell epimatrix and cancellous bone extracellular matrix, mucopolysaccharide, type i collagen, II type glue It is former.
A kind of above-mentioned syringeability cartilage takes off the preparation method of extracellular matrix mixing decalcified bone matrix hydrogel, packet Include following steps:
A prepares cartilage and takes off extracellular matrix freeze-drying sponge
1. impregnating of the same race or xenogenesis cartilage block with the Tris-HCl buffer containing phenylmethylsulfonyl fluoride, and cartilage is crushed, Distilled water repeated flushing；
2. the Tris-HCl buffer concussion 12 of X-100 containing Triton and phenylmethylsulfonyl fluoride~for 24 hours is added；
3. ultrapure water repeated flushing drains；
4. addition DNase I and RNase A concussion digestion 12~for 24 hours, ultrapure water repeated flushing；
5. being freeze-dried；
Sponge is lyophilized containing decalcified bone matrix in b preparation
1. impregnating of the same race or xenogenesis bone tissue with the Tris-HCl buffer containing phenylmethylsulfonyl fluoride, and by bone tissue powder It is broken, distilled water repeated flushing；
2. carrying out 12~72h of degreasing, distilled water repeated flushing with chloroform and methanol；
3. carrying out 12~72h of decalcification, distilled water repeated flushing with HCl solution；
4. CaCl is added2Solution stirs 1~3h, distilled water repeated flushing；EDTA solution is added and stirs 1~3h, distilled water Repeated flushing；LiCl solution is added and stirs 1~3h, distilled water repeated flushing；
5. the Tris-HCl buffer concussion 12 of X-100 containing Triton and phenylmethylsulfonyl fluoride~for 24 hours is added；
6. ultrapure water repeated flushing drains；
7. addition DNase I and RNase A concussion digestion 12~for 24 hours, ultrapure water repeated flushing；
8. being freeze-dried；
The preparation of c cartilage cell epimatrix mixing hydrogel containing decalcified bone matrix
1. the freeze-drying cartilage cell epimatrix obtained in mixing step a and step b according to a certain percentage and freeze-drying bone matrix, Pepsin is dissolved and be added with HCl solution, stirs 12~96h；
2. pH value of solution is adjusted to neutrality, 10 × PBS and 1 × PBS is added and mixes；
3. being placed 20~60 minutes under the conditions of 20~37 DEG C and being solidified into hydrogel.
The step a 1. with b 1. in cartilage and bone tissue be ground into 0~5mm × 0~5mm × 0~5mm size Grain.
1. 2. 1. 5. middle Tris-HCl buffer concentration is 0.01~0.1mol/L, pH7.4~7.5 to the step a with b； 1. 2. 1. 5. middle phenylmethylsulfonyl fluoride concentration is 0.035~0.35 mmol/L to step a with b；Step a 2. with b 5. in Triton X-100 concentration is 0.1~1 mmol/L；2. the volume ratio of middle chloroform and methanol is 1:1~2:1 to step b；3. middle HCl is dense by step b Spend 0.01~0.5 mol/L；Step b 4. in CaCl2The concentration of solution is 2~3.6mol/L, and the concentration of EDTA solution is 0.01 The concentration of~0.5mol/L, LiCl solution is 2~8mol/L；4. 7. middle DNase I and RNase A concentration is respectively step a with b 10~50 U/ml, 1~5 U/ml.
1. middle HCl concentration is 0.01~0.5 mol/L to the step c；Pepsin: cartilage cell epimatrix is freeze-dried mixed Bone matrix=50~150mg:1g；Step c 2. in adjustment pH value to neutral 0.01~1mol/L of NaOH concentration.
1. the mass ratio of middle cartilage cell epimatrix and freeze-drying bone matrix is less than 1:1 to the step c.
1. the mass ratio of middle cartilage cell epimatrix and freeze-drying bone matrix is 1:2 to the step c.
Step a, b carries out under the conditions of 0-4 DEG C in addition to digestion；2. step c is carried out under the conditions of 0~4 DEG C.
Present invention obtains following beneficial effects.
In the present invention, the II Collagen Type VI that cartilage takes off in the cartilage extracellular matrix obtained after cell can fill between inducing bone marrow Matter stem cell (BMSCs) differentiating cartilage-forming cell simultaneously has the characteristics that cartilage cell, and II Collagen Type VI can be in 37 DEG C of conditions Lower formation hydrogel has syringeability, good biodegradability and biocompatibility.Cancellous bone by decalcification, degreasing, The processing such as de- cell eliminate most non-collagens in addition to self-bone grafting forms albumen (BMP), greatly reduce bone and repair The antigenicity of multiple material, and there is good histocompatbility and degradability.Its hydrogel state has natural grid three Pore structure is tieed up, there is certain elasticity, syringeability is strong, and forms albumen containing self-bone grafting, can support and Induction of chondrocytes Plantation and proliferation.Cartilage extracellular matrix and cancellous bone extracellular matrix both natural cell epimatrix materials are combined water Gel, can be used as the bracket of compound cartilage cell in soft tissue engineering, and two kinds of extracellular matrix materials have complementary advantages, can make up It is respectively individually insufficient.Cancellous bone extracellular matrix contains BMP-2, has facilitation, aminoglucose to the differentiation of cartilage cell Polysaccharide (GAG) and II Collagen Type VI are that cartilage cell secretes generation, its secretion level can be improved in BMP-2.The present invention takes Material is simple, easy to operate, at low cost, can be used for cartilage defect filling and the treatment of minimally invasive orthopaedics repair of cartilage, has good face Bed application prospect.
Detailed description of the invention
Fig. 1 is the main view that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel；
Fig. 2 is the side view that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel；
Fig. 3 is the main view that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel freeze-drying sponge Figure；
Fig. 4 is the vertical view that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel freeze-drying sponge Figure；
Fig. 5 is that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel freeze-drying substantially microscope The mirror following figure；
Fig. 6 be syringeability cartilage of the present invention take off extracellular matrix mixing decalcified bone matrix hydrogel scanning electron microscope (SEM) photograph (× 500)；
Fig. 7 be syringeability cartilage of the present invention take off extracellular matrix mixing decalcified bone matrix hydrogel HE colored graph (× 100)；
Fig. 8 is that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel sarranine " O " colored graph (× 100)；
Fig. 9 is that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel Toluidine blue staining figure (× 100)；
Figure 10 is that syringeability cartilage of the present invention takes off extracellular matrix mixing decalcified bone matrix hydrogel type i collagen immune group Change figure；
Figure 11 is that the de- extracellular matrix mixing decalcified bone matrix hydrogel II Collagen Type VI of syringeability cartilage of the present invention is immune Groupization figure.
Specific embodiment
The present invention is described further with reference to the accompanying drawings and embodiments.
A kind of syringeability cartilage of the invention takes off the preparation method of extracellular matrix mixing decalcified bone matrix hydrogel such as Under:
A prepares cartilage and takes off extracellular matrix freeze-drying sponge (following operation is in addition to digestion under the conditions of 0~4 DEG C)
Fresh pig hyaline cartilage is taken, pH7.4 is 1. used, the Tris-HCl buffer of concentration 0.01mol/L (contains 0.035 Mmol/L phenylmethylsulfonyl fluoride) hyaline cartilage block is impregnated, 5mm × 5mm is made in cartilage with pulverizer (BILON, model DS-1) × 5mm size particles shape, distilled water repeated flushing；
2. the Tris-HCl buffering containing 1 mmol/L Triton X-100 and 0.035 mmol/L phenylmethylsulfonyl fluoride is added Liquid concussion 12~for 24 hours；
3. ultrapure water repeated flushing drains；
4. 50U/ml DNase I and 5U/mlRNase A is added shakes digestion 12h, ultrapure water repeated flushing at 37 DEG C；
5. being freeze-dried, -20 DEG C are saved backup.
B preparation is containing decalcified bone matrix freeze-drying sponge (following operation is in addition to digestion under the conditions of 0~4 DEG C)
Fresh porcine cancellous bone is taken, pH7.4 is 1. used, the Tris-HCl buffer of 0.01mol/L (contains 0.035 mmol/L benzene first Base sulfuryl fluoride) spongiosa bone block is impregnated, 5mm × 5mm × 5mm size is made in cancellous bone with pulverizer (BILON, model DS-1) It is granular, distilled water repeated flushing；
2. with chloroform: methanol=1:1 mixed liquor 12~72h of degreasing, distilled water repeated flushing；
3. with 0.01mol/L HCl solution (25~30ml/g bone) 12~72h of decalcification, distilled water repeated flushing；
4. with 2mol/L CaCl2Stir 1h, distilled water repeated flushing；0.5mol/L EDTA stirs 1h, and distilled water is repeatedly It rinses；8mol/L LiCl stirs 1h, distilled water repeated flushing；
5. the Tris-HCl buffering containing 1 mmol/L Triton X-100 and 0.035 mmol/L phenylmethylsulfonyl fluoride is added Liquid concussion 12~for 24 hours；
6. ultrapure water repeated flushing drains；
7. 50U/ml DNase I and 5U/ml RNase A is added shakes digestion 12h at 37 DEG C, ultrapure water rushes repeatedly It washes；
8. being freeze-dried, -20 DEG C are saved backup；
The preparation of c cartilage cell epimatrix mixing hydrogel containing decalcified bone matrix
1. taking 0.5g cartilage cell epimatrix mixing 1g that sponge is lyophilized containing decalcified bone matrix, it is dissolved in 20ml 0.01mol/L Pepsin 150mg is added in HCl solution, and stirring is for 24 hours；
2. under the conditions of 4 DEG C, taking 0.5ml cell mixing epimatrix, pH value is adjusted to neutrality with 0.1mol/L NaOH, is added 1 × PBS of 0.11ml 10 × PBS and 0.29ml is mixed；
3. placing 20 minutes at 37 DEG C, hydrogel can be solidified into.
Fig. 1,2 cartilage to be prepared using the method for the invention take off extracellular matrix mixing decalcified bone matrix hydrogel, Hydrogel diameter 1cm, height 1cm, milky, surface is smooth to be moistened.
Fig. 3,4 cartilage to be prepared using the method for the invention take off extracellular matrix mixing decalcified bone matrix hydrogel Dried frozen aquatic products, appearance off-white color is flexible, similar sponge.
Fig. 5 is that the cartilage prepared using the method for the invention takes off the jelly of extracellular matrix mixing decalcified bone matrix hydrogel It is seen under dry product substantially microscope, pore-creating character is good, similar sponge.
Fig. 6 scanning electron microscope (SEM) photograph, which is shown, takes off extracellular matrix mixing decalcification bone base using cartilage prepared by the method for the invention Matter hydrogel is rich in gap, and hole intercommunication.
Fig. 7 shows that taking off extracellular matrix mixing decalcified bone matrix hydrogel using cartilage prepared by the method for the invention revives Wood and Yihong (HE) dyeing have no carles indigo root dye nucleus (confirm that de- cell is thorough, cell is the main source of immunogenicity, this The rejection of sample hydrogel is substantially reduced), the extracellular matrix of visible eosin stains.
Fig. 8, which is shown, takes off extracellular matrix mixing decalcified bone matrix hydrogel kind using cartilage prepared by the method for the invention Red " O " dyeing: the proteoglycan (GAG) of visible stain.
Fig. 9, which is shown, takes off extracellular matrix mixing decalcified bone matrix hydrogel first using cartilage prepared by the method for the invention Aniline blue dyeing display staining for collagen is positive.
Figure 10, which is shown, takes off extracellular matrix mixing decalcified bone matrix hydrogel using cartilage prepared by the method for the invention Immunohistochemistry Type I collagen is positive.
Figure 11, which is shown, takes off extracellular matrix mixing decalcified bone matrix hydrogel using cartilage prepared by the method for the invention II Collagen Type VI of immunohistochemistry is positive.

Claims (9)

1. a kind of syringeability cartilage takes off the preparation method of extracellular matrix mixing decalcified bone matrix hydrogel, the hydrogel is By of the same race or xenogenesis cartilage by de- cell processing, of the same race or xenogenesis bone tissue is by degreasing, decalcification, de- cell processing, the two It is formed by after mixing according to a certain percentage and is dissolved under HCl solution, under conditions of 37 DEG C, neutral pH and suitable salinity Being solidified into has certain viscosity and the ossein hydrogel containing the osteogenic activity factor；It is characterized by comprising following steps:
A prepares cartilage and takes off extracellular matrix freeze-drying sponge
1. impregnating of the same race or xenogenesis cartilage block with the Tris-HCl buffer containing phenylmethylsulfonyl fluoride, and cartilage is crushed, is distilled Water repeated flushing；
2. the Tris-HCl buffer concussion 12 of X-100 containing Triton and phenylmethylsulfonyl fluoride~for 24 hours is added；
3. ultrapure water repeated flushing drains；
4. addition DNase I and RNase A concussion digestion 12~for 24 hours, ultrapure water repeated flushing；
5. being freeze-dried；
Sponge is lyophilized containing decalcified bone matrix in b preparation
1. impregnating of the same race or xenogenesis bone tissue with the Tris-HCl buffer containing phenylmethylsulfonyl fluoride, and bone tissue is crushed, is steamed Distilled water repeated flushing；
2. carrying out 12~72h of degreasing, distilled water repeated flushing with chloroform and methanol；
3. carrying out 12~72h of decalcification, distilled water repeated flushing with HCl solution；
4. CaCl is added2Solution stirs 1~3h, distilled water repeated flushing；EDTA solution is added and stirs 1~3h, distilled water rushes repeatedly It washes；LiCl solution is added and stirs 1~3h, distilled water repeated flushing；
5. the Tris-HCl buffer concussion 12 of X-100 containing Triton and phenylmethylsulfonyl fluoride~for 24 hours is added；
6. ultrapure water repeated flushing drains；
7. addition DNase I and RNase A concussion digestion 12~for 24 hours, ultrapure water repeated flushing；
8. being freeze-dried；
The preparation of c cartilage cell epimatrix mixing hydrogel containing decalcified bone matrix
1. the freeze-drying cartilage cell epimatrix obtained in mixing step a and step b according to a certain percentage and freeze-drying bone matrix, are used HCl solution dissolves and is added pepsin, stirs 12~96h；
2. pH value of solution is adjusted to neutrality, 10 × PBS and 1 × PBS is added and mixes；
3. being placed 20~60 minutes under the conditions of 37 DEG C and being solidified into hydrogel.
2. a kind of syringeability cartilage according to claim 1 takes off extracellular matrix mixing decalcified bone matrix hydrogel, Be characterized in that: described of the same race or xenogenesis cartilage and bone tissue are respectively derived from pig or ox or sheep or the cartilage and cancellous bone of people.
3. a kind of syringeability cartilage according to claim 2 takes off extracellular matrix mixing decalcified bone matrix hydrogel, Be characterized in that: the hydrogel contains cartilage cell epimatrix and cancellous bone extracellular matrix, mucopolysaccharide, type i collagen, II type glue It is former.
4. the system that a kind of syringeability cartilage according to claim 1 takes off extracellular matrix mixing decalcified bone matrix hydrogel Preparation Method, it is characterised in that: the step a 1. with b 1. in cartilage and bone tissue be ground into 0~5mm × 0~5mm × 0~ The particle of 5mm size.
5. the system that a kind of syringeability cartilage according to claim 1 takes off extracellular matrix mixing decalcified bone matrix hydrogel Preparation Method, it is characterised in that: 1. 2. 1. 5. middle Tris-HCl buffer concentration is 0.01~0.1mol/L to the step a with b, PH7.4~7.5；1. 2. 1. 5. middle phenylmethylsulfonyl fluoride concentration is 0.035~0.35mmol/L to step a with b；Step a 2. with b 5. Middle Triton X-100 concentration is 0.1~1mmol/L；2. the volume ratio of middle chloroform and methanol is 1:1~2:1 to step b；Step b 3. 0.01~0.5mol/L of middle HCl concentration；Step b 4. in CaCl2The concentration of solution be 2~3.6mol/L, EDTA solution it is dense Degree is 0.01~0.5mol/L, and the concentration of LiCl solution is 2~8mol/L；4. 7. middle DNase I and RNase A is dense with b by step a Degree is respectively 10~50U/ml, 1~5U/ml.
6. the system that a kind of syringeability cartilage according to claim 1 takes off extracellular matrix mixing decalcified bone matrix hydrogel Preparation Method, it is characterised in that: 1. middle HCl concentration is 0.01~0.5mol/L to the step c；Pepsin: the outer base of cartilage cell Freeze-dried mixed bone matrix=50 of matter~150mg:1g；Step c 2. in adjustment pH value to neutral 0.01~1mol/L of NaOH concentration.
7. the system that a kind of syringeability cartilage according to claim 1 takes off extracellular matrix mixing decalcified bone matrix hydrogel Preparation Method, it is characterised in that: 1. the mass ratio of middle cartilage cell epimatrix and freeze-drying bone matrix is less than 1:1 to the step c.
8. the system that a kind of syringeability cartilage according to claim 7 takes off extracellular matrix mixing decalcified bone matrix hydrogel Preparation Method, it is characterised in that: 1. the mass ratio of middle cartilage cell epimatrix and freeze-drying bone matrix is 1:2 to the step c.
9. the system that a kind of syringeability cartilage according to claim 1 takes off extracellular matrix mixing decalcified bone matrix hydrogel Preparation Method, it is characterised in that: step a, b carries out under the conditions of 0~4 DEG C in addition to digestion；Step c is 2. in 0~4 DEG C of condition Lower progress.
CN201610710756.4A 2016-08-24 2016-08-24 Syringeability cartilage takes off extracellular matrix mixing decalcified bone matrix hydrogel and preparation method thereof Active CN106075584B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610710756.4A CN106075584B (en) 2016-08-24 2016-08-24 Syringeability cartilage takes off extracellular matrix mixing decalcified bone matrix hydrogel and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610710756.4A CN106075584B (en) 2016-08-24 2016-08-24 Syringeability cartilage takes off extracellular matrix mixing decalcified bone matrix hydrogel and preparation method thereof

Publications (2)

Publication Number Publication Date
CN106075584A CN106075584A (en) 2016-11-09
CN106075584B true CN106075584B (en) 2019-07-05

Family

ID=57226169

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610710756.4A Active CN106075584B (en) 2016-08-24 2016-08-24 Syringeability cartilage takes off extracellular matrix mixing decalcified bone matrix hydrogel and preparation method thereof

Country Status (1)

Country Link
CN (1) CN106075584B (en)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR102018003726B1 (en) * 2018-02-26 2023-04-11 Universidade Federal Do Espírito Santo - Ufes PRODUCTION METHOD OF A BONE BIOMATERIAL, BONE BIOMATERIAL AND USE THEREOF
CN108744045A (en) * 2018-07-06 2018-11-06 宣城南巡智能科技有限公司 A kind of biocompatibility bone matrix of facial skeleton reparation injectable and preparation method thereof
CN111840642B (en) * 2020-07-15 2022-04-01 四川大学 Preparation method and application of cartilage acellular matrix composite scaffold
CN112316211A (en) * 2020-10-30 2021-02-05 南开大学 Cartilage extracellular matrix bionic injectable hydrogel and preparation method thereof
CN113456892A (en) * 2021-07-20 2021-10-01 中南大学湘雅医院 Method for preparing cartilage tissue engineering scaffold by using human umbilical cord Wharton jelly and cartilage tissue engineering scaffold
CN114796616B (en) * 2022-05-06 2023-10-10 广州医科大学附属第一医院（广州呼吸中心） Preparation method of hyaline cartilage acellular matrix composite scaffold
CN114984327B (en) * 2022-06-18 2023-04-25 广州医科大学 Composite bone defect filling agent and preparation method thereof
CN115444987B (en) * 2022-08-18 2023-07-11 江西中洪博元生物技术有限公司 Cartilage tissue decellularized hydrogel scaffold and preparation method and application thereof
CN116712609A (en) * 2023-07-26 2023-09-08 鑫华微(厦门)生物科技有限公司 Injectable temperature-sensitive composite hydrogel and application thereof in preparation of molding and/or repairing filling material

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000062834A1 (en) * 1999-04-20 2000-10-26 Schmidt K H Endoprosthesis with long-term stability
CN101574540A (en) * 2008-05-09 2009-11-11 中国人民解放军总医院 Tissue engineering bone/cartilage double-layer scaffold and construction method and application thereof
CN102552981A (en) * 2012-01-18 2012-07-11 北京申佑生物科技有限公司 Method for preparing tissue-engineered bone/cartilage integrated scaffold

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000062834A1 (en) * 1999-04-20 2000-10-26 Schmidt K H Endoprosthesis with long-term stability
CN101574540A (en) * 2008-05-09 2009-11-11 中国人民解放军总医院 Tissue engineering bone/cartilage double-layer scaffold and construction method and application thereof
CN102552981A (en) * 2012-01-18 2012-07-11 北京申佑生物科技有限公司 Method for preparing tissue-engineered bone/cartilage integrated scaffold

Also Published As

Publication number Publication date
CN106075584A (en) 2016-11-09

Similar Documents

Publication Publication Date Title
CN106075584B (en) Syringeability cartilage takes off extracellular matrix mixing decalcified bone matrix hydrogel and preparation method thereof
US11633518B2 (en) Graft scaffold for cartilage repair and process for making same
US9782517B2 (en) Crosslinked hyaluronic acid-collagen gels for improving tissue graft viability and soft tissue augmentation
CN108310467B (en) Assembled cell-derived extracellular matrix membrane composite bone repair material and preparation method and application thereof
JP6522520B2 (en) Solid substrate for promoting cell and tissue growth
ES2953309T3 (en) Injectable silk fibroin particles and uses thereof
US8546529B2 (en) Injectable bone regeneration gel containing bone formation enhancing peptide
Zhao et al. Osteogenic media and rhBMP-2-induced differentiation of umbilical cord mesenchymal stem cells encapsulated in alginate microbeads and integrated in an injectable calcium phosphate-chitosan fibrous scaffold
JP2015163234A (en) Solid form for tissue repairing
CN101564553B (en) Humanization active forging bone and preparation method thereof
US20130129835A1 (en) Crosslinked hyaluronic acid-collagen gels for improving tissue graft viability and soft tissue augmentation
JP2013533093A (en) Regenerative tissue scaffold
CN105246495A (en) Decellularized biomaterial form non-mammalian tissue
JP2021500091A (en) Adipose tissue products and manufacturing methods
CN106178119A (en) Syringeability decalcified bone matrix hydrogel and preparation method thereof
JP2021514820A (en) Decellularized Bone Extracellular Matrix Hydrogel-enhanced Decellularized Bone Biomaterial
Safdari et al. Recent advancements in decellularized matrix technology for bone tissue engineering
JP3646167B2 (en) Composite biomaterials containing phosphophorin
Haghwerdi et al. Application of bone and cartilage extracellular matrices in articular cartilage regeneration
EP2241606B1 (en) A method for preparing a biomaterial
CN105311676A (en) Hard tissue engineering scaffold material having bioactivity and preparation method of hard tissue engineering scaffold material
Young et al. Tissue engineering in oral and maxillofacial surgery
CN110624134A (en) I type/III type collagen cartilage repair bracket loaded with pilose antler polypeptide
Zhao et al. The application of polysaccharide biocomposites to repair cartilage defects
McCullen et al. Development and application of naturally renewable scaffold materials for bone tissue engineering

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant