CN105749348A - Preparation method of PRP (platelet-rich plasma)/MSCs (marrow stromal cells)/oyster shell bone tissue scaffold material - Google Patents
Preparation method of PRP (platelet-rich plasma)/MSCs (marrow stromal cells)/oyster shell bone tissue scaffold material Download PDFInfo
- Publication number
- CN105749348A CN105749348A CN201610135533.XA CN201610135533A CN105749348A CN 105749348 A CN105749348 A CN 105749348A CN 201610135533 A CN201610135533 A CN 201610135533A CN 105749348 A CN105749348 A CN 105749348A
- Authority
- CN
- China
- Prior art keywords
- oyster shell
- prp
- mscs
- bone
- preparation
- 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.)
- Pending
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
-
- 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
- A61L27/3616—Blood, e.g. platelet-rich plasma
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem 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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
-
- 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
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Transplantation (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Dermatology (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Urology & Nephrology (AREA)
- Botany (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Developmental Biology & Embryology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Disclosed is a preparation method of a PRP (platelet-rich plasma)/MSCs (marrow stromal cells)/oyster shell bone tissue scaffold material.A tissue engineering bone is constructed by taking oyster calcium carbonate as a scaffold, MSCs as seed cells and a PRP as an endogenous growth factor.The preparation method is simple and convenient.The tissue engineering bone, constructed by taking oyster shell as a scaffold, the marrow stromal cells as the seed cells and the platelet-rich plasma as the endogenous growth factor, is excellent in bone defect repairing effect and is similar to an autogenous bone graft, thereby being a satisfactory bone graft substitute material.A membrane-scaffold complex formed by wrapping the oyster shell scaffold by a membrane has excellent physical, chemical and mechanical properties, and is excellent in ectopic bone formation capability and biocompatibility, good in adhesive capacity for cells, capable of keeping normal growth, differentiation, proliferation and synthesizing and secreting functions and proper in biodegradation rate, and the materials and degradation products thereof have no toxic and side effect on organisms.
Description
Technical field
The present invention relates to organization material field, be specifically related to the system of a kind of PRP/MSCs/ oyster shell tissue scaffold design material
Preparation Method.
Background technology
Along with traffic accident and Patients with Bone Tumor increase and that big segmental defects self is repaired is impossible, bone collection
Quantity be only second to blood transfusion and occupied second place of the world.Because wound, particularly High energy trauma, joint replacement revision procedure,
The reasons such as excision of bone tumor cause bulk Cranial defect, need the patient carrying out bone collection to be on the increase, clinically to bone collection
The demand of material is increasing.Relevant statistics shows, the sale of global orthopaedics product in 2010 nearly 15,000,000,000 dollars.Right
The demand of surgical implant is increased sharply year by year.Autologous bone is undoubtedly optimal selection, but there is also all drawbacks simultaneously: (1) is normal
Limbs destroyed；(2) time and the danger of operation are increased；(3) convalescence of patient extends；(4) bone source is limited.(5) may be used
Can occur for the intrinsic complication such as district's pain, outward appearance is not good enough, local infection;Homogeneous allogenic bone, bone-xenograft source deficiency, can
Induction host produces immunological rejection, bone solution absorption, poor growth, transmission, increase POI probability and multiple
Miscellaneous ethics morals problem；The Ca-P ceramic class material such as hydroxyapatite, calcium phosphate bone cement has three dimensional pore structures, bone parent
Strong with power, but fragility is big, degraded is the most difficult.Therefore, find a kind of new tissue scaffold design material and have become as field of orthopaedics
Study hotspot.
Preferably artificial bone should have good biocompatibility, can effectively serve as the support of new bone formation, and energy
The most gradually degrade, substituted by bone tissue, the most also should have induction adjacent tissue mesenchymal cell and be divided into skeletonization
Cell or stimulating osteoblast accelerate a big class factor of propagation.It should have the best biocompatibility.2. to cell
Good adhesive capacity.3. can keep the normal growth of cell, break up, breed and synthesis secretion function.4. it is suitable to have
Biodegradation rate.5. body is had no side effect by material and catabolite thereof.6. there is good physics, chemistry and mechanicalness
Energy.7. can sterilize.Find most attraction that preferable implantation always orthopedist faces and challenging
Problem.In a word, the general orientation of implantation research is minimum autologous wound；The most imitative autologous bone, not
The form of the nearly autologous bone of disconnecting, 26S Proteasome Structure and Function.
Platelet rich plasma (platelet-rich plasma, PRP) is to be extracted from autologous blood by centrifugal method
Platelet concentrate out, rich in various growth factors.PRP is initially applied to as a kind of biogel and barrier film
Clinic, primarily serves the purpose of raising haemostatic effect.From Assoian in 1984 find in PRP containing multiple growth factor (as
PDGF:platelet-derived growth factor platelet derived growth factor, TGF-b1, TGF-b2:
Transforming growth factor growth factor has Peritoneal fibrosis b1, b2, and IGFs: insulin-like
Growth factors IGF, VEGF:vascular endothelial growth factor Ink vessel transfusing
Skin cell growth factor, EGF:epidermal growth factor EGF), application constantly expands thereafter.
After Marx in 1998 etc. confirm that PRP has promotion osteogenic action, many scholars are around PRP application start in Bone Defect Repari
Substantial amounts of research, and achieve certain achievement.PRP can promote Bone Defect Repari to be primarily due to wherein to contain a large amount of osteanagenesis
Required growth factor, and the ratio of each growth factor is body self formation, and it is numerous that this characteristic causes orthopedics circle
The concern of scholar.
Oyster shell calcium: 1. oyster shell has preferable voidage, porosity is up to about 40-60%.2. oyster housing
It is to be constituted 3. cross section by nearly nano particle by dispersion-strengtherning and plate object staggered accumulation strengthening to can observe 5～20 layers of bone
Plate spline structure, arranges in concentric column, and plate thickness, between 200-300nm, has the pipe-line system communicated with each other to be connected between plate
System, the widest pipeline reaches 600nm, averagely between 300nm, this and the hone lamella of human bone, Haversian canal and floating gram graceful pipe
Quantity, density, shape, aperture, to move towards rule similar.Oyster shell material hole is abundant, aperture is suitable, moderate strength, every
Physicochemical property is excellent;The biocompatibility of oyster shell material is good, can occur biodegradable in vivo;Oyster shell material has
Osteoconduction ability, is a kind of promising bone alternate material.
Marrow stromal cell (marrow stromal cells, MSCs) 1. has many differentiation potentials, 2. at specific bar
Can break up to Gegenbaur's cell, cartilage cell and adipocyte etc. under part, 3. multiplication capacity is strong, 4. wide material sources, 5. draws materials conveniently,
Little for district's damage is one of comparatively ideal seed cell of bone tissue engineer.
Mount in 2004 etc. have with bone mineralization in coral formation and human body in the formation of Science report oyster shell
High similarity, research uses the coastal oyster in Asia, it was demonstrated that the formation of oyster shell is also the inorganic salts constantly secreted by granulocyte
The mineral salt piled up and formed.Oyster shell contains abundant trace element and a large amount of amino acid.The earliest about oyster as life
The report of thing material is in 1965, and research does not has any bad reaction in showing to be implanted into Mice Body.Using oyster as
Original material[18]Being processed into oyster shell calcium calcium tonic to ratify through U.S. FDA in 1997, within 2002, oyster calcium obtains state
The Ministry of Public Health of family lot number.The intensity data that Currey J D etc. measure oyster shell is suitable with the cortex bone of ox in document[19], far away
Higher than Porites coral, illustrate that it has preferable cortex bone and substitutes potential.Oyster shell particulate component: CaCO3 (95.994 %)
SiO2 (0.696%) MgO(0.649 %) Al2O3 (0.419%) SrO (0.33%) P2O5 (0.204%) Na2O
(0.984%) SO3(0.724%)[20]。
Marine biology combines with biotechnology, creates this new field of marine biotechnology.Many countries are
Using marine biotechnology as the important component part of 21st century development strategy.The Optimum utilization of living marine resources and high level
Change processing is one of important research content of China's Marine High-technology development in following 15 years.In the U.S., most bones is applied to move
Planting material is coral hydroxyapatite (CHA), and it is similar to cancellous bone structure, porous, biocompatibility are preferable.In organizational project
In the research of bone, the most a large amount of use South Sea Porites coral, cuttle bone all achieve preferably as engineering material of bone tissue, experiment
Bone Defect Repari effect.But, above-mentioned material also exists poor plasticity, and intensity is difficult to reach the requirement of big section bone alternate material, institute
By defects such as raw material Coral state guarantee preciousness natural resources, limited amounts.Therefore, it is necessary to develop new marine organisms
Bone alternate material.
Summary of the invention
In order to solve the shortcoming of prior art, the present invention provides a kind of PRP/MSCs/ oyster shell tissue scaffold design material
Preparation method.
The technical solution that the present invention uses is: the preparation side of a kind of PRP/MSCs/ oyster shell tissue scaffold design material
Method, comprises the following steps:
(1) oyster shell supporting frame prefabrication is standby: take oyster shell, washes away air-dried, is rolled by oyster shell and is crushed to graininess, first with after 40 mesh
Filter with 100 mesh sieves, obtain 100 mesh particles below, then ostreae testa pulverata is placed in 10% chloroformic solution immersion
24h, seals, and stirs, and uses distilled water flushing 3 times after 24h, filters, and is then added by ostreae testa pulverata in 30% hydrogen peroxide and soaks
24h, and stirring, thereafter distilled water immersion, rinse, filter, 3 times repeatedly, last natural air drying, oyster shell powder is immersed in aseptic
In physiological saline, remove the impurity in oyster shell powder with ultrasonic numerical control machine, and be dried under 37 DEG C of constant temperatures, seal and preserve；
(2) preparation of oyster shell support: at sonic oscillation, under conditions of 40 DEG C, adds absolute ethyl alcohol, to anhydrous in agitator
Adding oyster shell powder and α-half-H 2 O calcium sulphate with 1:3 mass ratio in ethanol, after vibration 20min, both mix and make α-half water
Calcium sulfate is uniformly distributed in oyster shell powder, then injects the mixture in mould, and mould is put room temperature, 4 DEG C of gradient falls successively
The each 1h of temperature, under the conditions of 4 DEG C overnight, is lyophilized 72 hours under 500Pa negative pressure, obtains oyster shell support；
(3) preparation of thrombin of beef solution: under aseptic condition, 5000U thrombin of beef is added aseptic 10% calcium chloride of 5ml
In solution, shake up, standby；
(4) preparation of platelet rich plasma: under aseptic condition, with the 10ml note being pre-loaded with 2ml 10% sodium citrate anticoagulant
Emitter, extracts 8ml blood in Rabbit central ear artery, shakes up, insert in centrifuge tube, carries out twice and is centrifuged, the most centrifugal
200g × 10min, blood is divided into the blood plasma on upper strata and the red blood cell two-layer of lower floor, draws whole blood plasma and upper strata about 1 mm is high
The red blood cell of degree, in another centrifuge tube, carries out the centrifugal 250g × 10min of second time, and blood plasma is divided into the weary blood platelet blood on upper strata
Slurry (PPP) and the PRP of lower floor, discard the PPP on upper strata, remains 1.5ml liquid, shakes up, obtain PRP, PRP is stored in-70 DEG C
In condition standby；
(5) rabbit MSCs separation, cultivate, expand and induce: after 0.1ml/kg intramuscular anesthesia rabbit, cut under aseptic condition
Both sides ilium, is put into filling in the bottle of 10ml DMEM complete culture solution, shreds, and piping and druming makes marrow fully separate out, so
After by liquid subpackage, move in culture dish, add above-mentioned DMEM complete culture solution, insert in incubator, saturated humidity, 37
Hatch under the conditions of C, 5%CO2, within second day, change liquid, remove non-attached cell and broken bone piece, within later every 2 days, change liquid 1 time, carefully
When born of the same parents cover with 80%, 0.25% trypsinization, pass on, when second generation cell covers with to 80%, add the training of DMEM induction broth
Supporting, when cell length to 90-100%, with 0.25% trypsinization, centrifugal, counting, being prepared as cell concentration is 1.0 × 107/
The cell suspension of ml, standby；
(6) MSCs and PRP plantation on oyster shell support and in vitro culture: taking 50 μ l cell concentrations is 1.0 × 107/
The MSCs suspension of ml and the PRP mixing of the 50 same donor sources of μ l, shake up, and is added drop-wise on oyster shell disk by this mixing liquid,
Drip the thrombin of beef solution of 10 μ l again, form oyster shell/MSCs/PRP tissue scaffold design material.
Described step (1) obtains below 100 mesh ostreae testa pulverata particle use with after 40 mesh with 100 mesh sieves
Filter and obtain.
In described step (5) DMEM complete culture solution comprise 100ml/L hyclone, 50 mg/L ascorbic acid,
0.272 g/L Glu, 100 U/ml penicillin, 100 mg/ml streptomysins.
In described step (5) DMEM induction broth comprise 10mmol/L sodium β-glycerophosphate, 10-7Mol/L ground plug rice
Pine.
The invention has the beneficial effects as follows: the preparation method of a kind of PRP/MSCs/ oyster shell tissue scaffold design material, the present invention
To build organizational project as seed cell, PRP as Endogenous Growth Factors as support, MSCs using oyster shell calcium
Bone, employing oyster shell makees support, marrow stromal cell makees seed cell, platelet rich plasma is made Endogenous Growth Factors and built
Tissue Engineering Bone for Repair of Bone Defect respond well, close with autologous bone transplanting, be comparatively ideal bone transplantation substitute material, method
Simplicity, film-support complex that diaphragm parcel oyster shell support is formed has good ectopic bone formation, has good biology
Compatibility, the adhesive capacity good to cell, the normal growth of cell can be kept, break up, breed and synthesis secretion function, tool
There are suitable biodegradation rate, material and catabolite thereof that body is had no side effect, there is good physics, chemistry and machine
Tool performance.
Accompanying drawing explanation
Fig. 1 is the technology of the present invention route map.
Fig. 2 is oyster shell powder 0.15mm(X 400) micrograph.
Fig. 3 is the Making programme figure of oyster shell composite.
Fig. 4 is oyster shell scanning electron microscope (SEM) photograph.
Fig. 5 is bone material hole, hone lamella traffic, nearly nanoparticle structure Electronic Speculum figure.
Fig. 6 is Primary bone marrow stroma stem cell alkaline phosphatase staining (× 40) figure.
Fig. 7 is that cell cultivates the 2nd, 7,20 day SEM figure (× 2.5K) with Material cladding.
Fig. 8 is the present invention postoperative same day, 4,8,16,24 weeks and normal x line sheet.
Detailed description of the invention
Research contents:
1, PRP is on Marrow Stromal Cells in Proliferation on oyster shell support and the impact of Osteoblast Differentiation
From rabbit Iliac Bone, isolate MSCs, in vitro culture, expand and induce.Taking 50 μ l cell concentrations is 1.0 × 107/
The MSCs suspension of ml mixes with 50 μ l PRP of same donor source, is added drop-wise to diameter 8 mm, the oyster shell disk of thick 3 mm
On, then drip the thrombin of beef solution of 10 μ l, and form oyster shell/MSCs/PRP compound. at saturated humidity, 37 DEG C, 5%CO2
Under the conditions of cultivate.Oyster shell/MSCs the compound prepared with same amount of MSCs or PRP and oyster shell/PRP compound are at body
Outer cultivation compares.After 8 days and 14 days, by mtt assay detection PRP on the impact of MSCs propagation on oyster support；By right
Nutrient solution alkaline phosphatase (alkaline phosphatase, ALP) activity and BGP (osteocalcin, OC) contain
Amount detection, evaluates PRP to the impact of MSCs Osteoblast Differentiation on oyster support；By scanning electron microscopic observation MSCs on oyster support
Attachment, growth and proliferative conditions.
2, PRP is on the impact of marrow stromal cell ectopic osteogenesis on oyster shell support
From rabbit Iliac Bone, isolate MSCs, in vitro culture, expand and induce.Taking 50 μ l cell concentrations is 1.0 × 108/
The MSCs suspension of ml mixes with 50 μ l PRP of same donor source, is added drop-wise to diameter 8 mm, the oyster shell disk of thick 3 mm
On, then drip the thrombin of beef solution of 10 μ l, and form oyster shell/MSCs/PRP compound. with same amount of MSCs or PRP
Preparation oyster/MSCs compound and oyster/PRP compound.By oyster shell/MSCs/PRP compound, oyster/MSCs compound and
Oyster/PRP compound implants the dorsal sc of same nude mice, within postoperative 4 weeks and 8 weeks, draws materials.By gross examination of skeletal muscle, histology
Observe and Histomorphometric analysis evaluates its ectopic osteogenesis situation.
3, oyster shell/marrow stromal cell/PRP repairs femoral defects in rabbits experimental study
From rabbit Iliac Bone, isolate MSCs, in vitro culture, expand and induce.Taking 50 μ l cell concentrations is 1.0 × 108/
The MSCs suspension of ml mixes with 50 μ l PRP of same donor source, is added drop-wise to diameter 8 mm, the oyster shell disk of thick 3 mm
On, then drip the thrombin of beef solution of 10 μ l, and form oyster shell/MSCs/PRP compound. repair MSCs and PRP source with it
The diameter 10 mm femur defect of rabbit.Autologous femur or simple femur are implanted and are compared.Postoperative 6 weeks and 12 weeks, by greatly
Body observation, X-ray film observation, histological observation and Histomorphometric analysis, evaluate its Bone Defect Repari effect.
Goal in research
(1) checking PRP promotes Marrow Stromal Cells in Proliferation and Osteoblast Differentiation on oyster shell support.
(2) checking PRP promotes marrow stromal cell ectopic osteogenesis on oyster shell support.
(3) oyster shell/marrow stromal cell/PRP, oyster/MSCs and oyster/PRP biological support are built.
Preparation method
The preparation of oyster shell support:
(1) taking oyster shell, brush punching air-dries, and with medical pulverizer, oyster shell is rolled cause graininess, first uses 100 mesh with after 40 mesh
Sieves filters, and obtains 100 mesh particles below. and ostreae testa pulverata is placed in 10% chloroformic solution immersion 24h, seals, and use
Agitator stirs.Use distilled water flushing 3 times after 24h, filter.It is subsequently adding in 30% hydrogen peroxide immersion 24h, and uses agitator
Stirring.Thereafter distilled water immersion, rinse, filter, 3 times repeatedly, last natural air drying.Before using, oyster shell powder is immersed in aseptic
In physiological saline, remove the impurity in oyster shell powder with ultrasonic numerical control machine, and dry in the electric heating constant-temperature blowing drying box of 37 DEG C
Dry, seal before using and preserve.
(2) at sonic oscillation, under conditions of 40 DEG C, in magnetic stirring apparatus add absolute ethyl alcohol, in absolute ethyl alcohol with
1:3 mass ratio adds oyster shell powder and α-half-H 2 O calcium sulphate, and after vibration 20min, both mix and make α-half-H 2 O calcium sulphate equal
Even be distributed in oyster shell powder, then inject the mixture into 8mm * 3mm in molding jig, mould is put successively room temperature, 4 DEG C
The each 1h of gradient cooling, 4 DEG C of refrigerator overnight, it is lyophilized 72 hours under 500Pa negative pressure, cmposite artificial bone is given oxirane and disappears
Poison, seals up for safekeeping standby.
The preparation of thrombin of beef solution
Under aseptic condition, 5000U thrombin of beef is added in aseptic 10% calcium chloride solution of 5ml, shake up, standby.
The preparation of platelet rich plasma
Under aseptic condition, use the 10ml syringe being pre-loaded with 2ml 10% sodium citrate anticoagulant, in Rabbit central ear artery
Extraction 8ml blood, shakes up, inserts in centrifuge tube, carries out twice and is centrifuged.The most centrifugal 200g × 10min, blood is divided into
The blood plasma of layer and the red blood cell two-layer of lower floor.Draw the red blood cell of whole blood plasma and upper strata about 1 mm height to another centrifuge tube
In, carry out the centrifugal 250g × 10min of second time.Blood plasma is divided into platelet-poor plasma (the platelet poor on upper strata
Plasma, PPP) and the PRP of lower floor.The PPP(discarding upper strata accounts for the 70% of blood plasma cumulative volume), remaining liq about 1.5ml,
Shake up, be PRP[23]PRP is stored in-70 DEG C of refrigerators standby.
The separation of rabbit MSCs, cultivate, expand and induce
After Su Mian Xin intramuscular anesthesia (0.1ml/kg) new zealand rabbit, under aseptic condition, cut both sides ilium.In ultra-clean work
In platform, be put into filling 10ml DMEM complete culture solution (containing 100ml/L hyclone, 50 mg/L ascorbic acid,
0.272 g/L Glu, 100 U/ml penicillin, 100 mg/ml streptomysins) bottle in, shred, piping and druming, make bone
Marrow fully separates out, and then by liquid subpackage, immigration culture dish, adds above-mentioned DMEM complete culture solution, inserts in incubator,
Saturated humidity, 37 DEG C, hatch under the conditions of 5%CO2.Within second day, change liquid, remove non-attached cell and broken bone piece.The most every 2 days
Change liquid 1 time.When cell covers with 80%, 0.25% trypsinization, pass on.When second generation cell covers with to 80%, add DMEM
Induction broth (in complete culture solution add 10mmol/L sodium β-glycerophosphate, 10-7Mol/L dexamethasone) cultivate (figure
3).When cell length to 90-100%, with 0.25% trypsinization, centrifugal, counting, being prepared as cell concentration is 1.0 × 107/
The cell suspension of ml, standby[24]
MSCs and PRP plantation on oyster shell support and in vitro culture
Taking 50 μ l cell concentrations is 1.0 × 107The MSCs suspension of/ml and the PRP mixing of the 50 same donor sources of μ l, shake up.
This mixing liquid is added drop-wise on oyster shell disk, then drips the thrombin of beef solution of 10 μ l, form oyster shell/MSCs/PRP
Compound.As comparison, taking 50 μ l cell concentrations is 1.0 × 107The MSCs suspension of/ml is added drop-wise on oyster shell disk, shape
Become oyster shell/MSCs compound.Take 50 μ l PRP to be added drop-wise on oyster shell disk, then drip the thrombin of beef solution of 10 μ l,
Form oyster shell/PRP compound.Above-mentioned three kinds of compounds are respectively placed in 6 well culture plates, put into incubator, saturated wet
Degree, 37 DEG C, hatch 4h under the conditions of 5%CO2 after, add 5ml DMEM complete culture solution and continue to hatch, within every 2 days, change liquid 1
Secondary.
Testing index
ALP activity and OC assay in nutrient solution[25]: in vitro culture 8 days and 14 days, detection cell is discharged into nutrient solution
In ALP activity and OC content.Every time point often group takes 6 parts of samples, draws the nutrient solution 1ml in each hole of culture plate, is divided into
Two equal portions.Take a nutrient solution, operate by ALP kit specification, with spectrophotometric determination 405nm wavelength extinction
Degree, calculates ALP activity.Taking another part of nutrient solution, operate by BGP kit specification, machine of finally going up measures in sediment to be put
Penetrating property counts, and processes through RIA software and draws OC content.
Cell proliferating determining: in vitro culture 8 days and 14 days, oyster shell/MSCs/PRP compound and oyster shell/
In the culture hole of MSCs compound (often organizing 6 holes), each MTT 100 μ l adding 50g/L, continues to cultivate 4h, absorbs training
Nutrient solution, adds 500 μ l dimethyl sulfoxide (DMSO)s in every hole, vibrate 10min, surveys at 490 nm wavelength with enzyme-linked immunosorbent assay instrument
Determine OD value.
Scanning electron microscopic observation: in vitro culture is after 8 days and 14 days, often organizes and takes 3 samples respectively and be placed in 2.5% penta 2
Aldehyde solution is fixed, lyophilized, metal spraying, scanning electron microscopic observation.
PRP is on the impact of marrow stromal cell ectopic osteogenesis on oyster shell support
(1) rabbit MSCs and PRP plantation on oyster shell support:
Take cell suspension that 50 μ l cell concentrations are 1.0 × 108/ml and 50 μ l to mix from the PRP of same donor, shake up.
Mixture is added drop-wise on oyster shell disk, then drips the thrombin of beef solution of 10 μ l, form oyster shell/MSCs/PRP and be combined
Thing.As comparison, take 50 μ l cell suspensions and be added drop-wise on oyster shell disk, form oyster shell/MSCs compound.Take 50 μ l
PRP is added drop-wise on oyster shell disk, then drips the thrombin of beef solution of 10 μ l, forms oyster shell/PRP compound.
(2) BALB/c nude mice 16 is selected.Su Mian Xin intramuscular anesthesia (1ml/kg), under aseptic condition, at every
1cm skin incision is made at nude mice back, and blunt separation forms three subcutaneous pockets, implants oyster shell/MSCs/PRP, oyster respectively
Shell/MSCs and oyster shell/PRP compound, tightly sew up a wound.
(3) Testing index
Within postoperative 4 weeks and 8 weeks, put to death animal (each time point 8) respectively, draw materials.First carry out gross examination of skeletal muscle.Again will mark
Originally be placed in 10% neutral formalin liquid fixing, decalcification, dehydration, FFPE, indulge shape section from sample center, HE and
MassonShi trichrome stain, light Microscopic observation.Each sample takes three continuous print MassonShi trichrome at light microscopic
Under carry out into quantitative bone measurement by image analysis system.
Oyster shell/marrow stromal cell/PRP repairs femoral defects in rabbits experimental study
(1) rabbit MSCs and PRP plantation on oyster shell support
Take cell suspension that 300 μ l cell concentrations are 1.0 × 108/ml and 300 μ l to mix from the PRP of same donor, shake
Even.Mixture is added drop-wise on oyster shell disk, drips the thrombin of beef solution of 100 μ l, form oyster shell/MSCs/PRP multiple
Compound.Insert incubator, saturated humidity, 37 DEG C, under the conditions of 5%CO2, stand 1h, standby.
(2) 5% yellow Jackets 1ml/kg anaesthetize from auricular vein.The double hind leg shaving of animal, routine disinfection, drape, from
Before bilateral knee joint, outside is about the longitudinal incision of 2cm, appears distal part of femur；With ring electric drill at knee joint 1cm, cause 1 ×
The Cranial defect of 1cm size.Experimental group femoral defects in rabbits be implanted into the oyster shell prepared by autogenous MSCs and PRP/
MSCs/PRP tissue engineered bone, tightly sews up a wound.According to the method described above, at positive controls and negative control group femur
Implant autologous femur and simple oyster shell support, animal sub-cage rearing in defect respectively, do not do any specially treated.
(3) observation index
Within postoperative 6 weeks and 12 weeks, often organizing 6 animals of each execution, intercepting implant and about 2mm normal bone tissues are carried out
Following observation: 1. gross examination of skeletal muscle, removes sample surrounding soft tissue, visually observes Cranial defect district bon e formation situation, and use clamp side
Method compares the difference in hardness in defect repair district and periphery sclerotin.2. x-ray takes the photograph sheet observation, in voltage 50V, electric current 100MA, exposure
Taking the photograph sheet under the exposure factor of time 0.05s, situation is penetrated in the x-ray resistance observing defect repair district.3. histological observation, sample is normal
Regulation becomes continuous decalcification paraffin section, and HE dyes, light Microscopic observation.4. graphical analysis, with image analysis system to histotomy
Carry out into quantitative bone measurement, become bone amount to represent with the bone forming area (μm 2) in section；Choosing is complete through material center, tangent plane
Histotomy 2, counts mean value, to represent the one-tenth bone amount of this sample；Institute's value is carried out statistical procedures.
The preparation of RP is simple, through centrifugal just can obtain hematoblastic concentrate i.e. after extracting blood out from vein or artery
PRP.Now with the multiple commercially available seperator quickly preparing PRP, PRP is easier, quick in preparation.U.S. FDA approval clinic
2 kinds of PRP seperator Smart PreP and PCCS of application, can prepare containing high concentration blood platelet and a large amount of active growth
The PRP of the factor[26]。
Blood platelet is derived from autologous, will not cause the infectious diseases such as hepatitis, AIDS and rabid ox disease.Wound is little, it is easy to quilt
Patient accepts.Whether application thrombin of beef can cause rabid ox disease as the coagulant of PRP？In terms of clinical practice, thrombin of beef is
It is applied to the patient of the surgery every field of more than 1,000 ten thousand, does not all occur this sick.Saying, rabid ox disease is by albumen sense again
Contaminate what plain this propagation medium was propagated.Prion exists only in neural group of the central nervous system of ox, sheep, cat and people etc.
In knitting, and thrombin of beef is by extracting in blood, and through hot-working and purifying, thus be safe.
Recently, U.S. FDA eliminates original recombinant human bone morphorgenetic protein-2 and can be applicable to cranio-facial surgery and oral and maxillofacial surgery
The approval of surgical clinical.Because recombinant human bone morphorgenetic protein-2 may cause bone tumour.Therefore, PRP is as the most endogenous
Property growth factor source just seem even more important, have broad application prospects[27]。
This project system is standby to be gone out to have the organizational project bone grafting material of high bioactivity, controlled degradation, has the strongest innovation
Property, have independent intellectual property right, can be used for the reparation of clinical Cranial defect, the necks such as domestic biomaterial and tissue engineered bone will be promoted
The technology development and progress in territory, promotes our province economy and the development of related industry, will bring great social profit and economic effect
Benefit.
Experiment conclusion
1) platelet rich plasma promotes propagation and the skeletonization of marrow stromal cell on the oyster shell support of in vitro culture
Differentiation.
2) platelet rich plasma promotes the ectopic osteogenesis of marrow stromal cell on oyster shell support.
3) with oyster shell make support, marrow stromal cell makees seed cell, platelet rich plasma makees Endogenous Growth Factors
The Tissue Engineering Bone for Repair of Bone Defect built is respond well, close with autologous bone transplanting, is comparatively ideal bone transplantation substitute material.
4) the method simplicity of organizational project diaphragm, diaphragm parcel oyster is built with platelet rich plasma and marrow stromal cell
Film-support complex that shell support is formed has good ectopic bone formation.
Claims (4)
1. the preparation method of a PRP/MSCs/ oyster shell tissue scaffold design material, it is characterised in that comprise the following steps:
(1) oyster shell supporting frame prefabrication is standby: take oyster shell, washes away air-dried, is rolled by oyster shell and is crushed to graininess, first with after 40 mesh
Filter with 100 mesh sieves, obtain 100 mesh particles below, then ostreae testa pulverata is placed in 10% chloroformic solution immersion
24h, seals, and stirs, and uses distilled water flushing 3 times after 24h, filters, and is then added by ostreae testa pulverata in 30% hydrogen peroxide and soaks
24h, and stirring, thereafter distilled water immersion, rinse, filter, 3 times repeatedly, last natural air drying, oyster shell powder is immersed in aseptic
In physiological saline, remove the impurity in oyster shell powder with ultrasonic numerical control machine, and be dried under 37 DEG C of constant temperatures, seal and preserve；
(2) preparation of oyster shell support: at sonic oscillation, under conditions of 40 DEG C, adds absolute ethyl alcohol, to anhydrous in agitator
Adding oyster shell powder and α-half-H 2 O calcium sulphate with 1:3 mass ratio in ethanol, after vibration 20min, both mix and make α-half water
Calcium sulfate is uniformly distributed in oyster shell powder, then injects the mixture in mould, and mould is put room temperature, 4 DEG C of gradient falls successively
The each 1h of temperature, under the conditions of 4 DEG C overnight, is lyophilized 72 hours under 500Pa negative pressure, obtains oyster shell support；
(3) preparation of thrombin of beef solution: under aseptic condition, 5000U thrombin of beef is added aseptic 10% calcium chloride of 5ml
In solution, shake up, standby；
(4) preparation of platelet rich plasma: under aseptic condition, with the 10ml note being pre-loaded with 2ml 10% sodium citrate anticoagulant
Emitter, extracts 8ml blood in Rabbit central ear artery, shakes up, insert in centrifuge tube, carries out twice and is centrifuged, the most centrifugal
200g × 10min, blood is divided into the blood plasma on upper strata and the red blood cell two-layer of lower floor, draws whole blood plasma and upper strata about 1 mm is high
The red blood cell of degree, in another centrifuge tube, carries out the centrifugal 250g × 10min of second time, and blood plasma is divided into the weary blood platelet blood on upper strata
Slurry (PPP) and the PRP of lower floor, discard the PPP on upper strata, remains 1.5ml liquid, shakes up, obtain PRP, PRP is stored in-70 DEG C
In condition standby；
(5) rabbit MSCs separation, cultivate, expand and induce: after 0.1ml/kg intramuscular anesthesia rabbit, cut under aseptic condition
Both sides ilium, is put into filling in the bottle of 10ml DMEM complete culture solution, shreds, and piping and druming makes marrow fully separate out, so
After by liquid subpackage, move in culture dish, add above-mentioned DMEM complete culture solution, insert in incubator, saturated humidity, 37
Hatch under the conditions of C, 5%CO2, within second day, change liquid, remove non-attached cell and broken bone piece, within later every 2 days, change liquid 1 time, carefully
When born of the same parents cover with 80%, 0.25% trypsinization, pass on, when second generation cell covers with to 80%, add the training of DMEM induction broth
Supporting, when cell length to 90-100%, with 0.25% trypsinization, centrifugal, counting, being prepared as cell concentration is 1.0 × 107/
The cell suspension of ml, standby；
(6) MSCs and PRP plantation on oyster shell support and in vitro culture: taking 50 μ l cell concentrations is 1.0 × 107/
The MSCs suspension of ml and the PRP mixing of the 50 same donor sources of μ l, shake up, and is added drop-wise on oyster shell disk by this mixing liquid,
Drip the thrombin of beef solution of 10 μ l again, form oyster shell/MSCs/PRP tissue scaffold design material.
The preparation method of a kind of PRP/MSCs/ oyster shell tissue scaffold design material the most according to claim 1, its feature exists
In, the ostreae testa pulverata particle employing obtaining below 100 mesh in described step (1) obtains with 100 mesh sieves filtrations with after 40 mesh
?.
The preparation method of a kind of PRP/MSCs/ oyster shell tissue scaffold design material the most according to claim 1, its feature exists
In, in described step (5), DMEM complete culture solution comprises 100ml/L hyclone, 50 mg/L ascorbic acid, 0.272 g/
L Glu, 100 U/ml penicillin, 100 mg/ml streptomysins.
The preparation method of a kind of PRP/MSCs/ oyster shell tissue scaffold design material the most according to claim 1, its feature exists
In, in described step (5) DMEM induction broth comprise 10mmol/L-sodium glycero-phosphate, 10-7Mol/L dexamethasone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610135533.XA CN105749348A (en) | 2016-03-10 | 2016-03-10 | Preparation method of PRP (platelet-rich plasma)/MSCs (marrow stromal cells)/oyster shell bone tissue scaffold material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610135533.XA CN105749348A (en) | 2016-03-10 | 2016-03-10 | Preparation method of PRP (platelet-rich plasma)/MSCs (marrow stromal cells)/oyster shell bone tissue scaffold material |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105749348A true CN105749348A (en) | 2016-07-13 |
Family
ID=56333023
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610135533.XA Pending CN105749348A (en) | 2016-03-10 | 2016-03-10 | Preparation method of PRP (platelet-rich plasma)/MSCs (marrow stromal cells)/oyster shell bone tissue scaffold material |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105749348A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106362213A (en) * | 2016-11-02 | 2017-02-01 | 中国人民解放军第三军医大学第三附属医院 | PRP loaded slow release antibiotic-mesenchymal stem cell gel system and preparation method thereof |
| CN108187133A (en) * | 2018-01-09 | 2018-06-22 | 楼毅 | A kind of preparation method of medical composite type α-half-H 2 O calcium sulphate artificial bone |
| CN110694114A (en) * | 2019-10-31 | 2020-01-17 | 南通大学附属医院 | PRP-loaded pancreas acellular scaffold and preparation method thereof |
| CN112717204A (en) * | 2021-01-19 | 2021-04-30 | 郭燕庆 | Autologous bone graft activity substitute composition, preparation method and application |
| CN112791236A (en) * | 2020-12-18 | 2021-05-14 | 广东普罗凯融生物医药科技有限公司 | Cell microcarrier matrix and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2824875Y (en) * | 2005-04-14 | 2006-10-11 | 南方医院 | An injection type tissue engineering bone engraft |
-
2016
- 2016-03-10 CN CN201610135533.XA patent/CN105749348A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2824875Y (en) * | 2005-04-14 | 2006-10-11 | 南方医院 | An injection type tissue engineering bone engraft |
Non-Patent Citations (8)
| Title |
|---|
| YUE SHEN ET AL.: "Engineering Scaffolds Integrated with Calcium Sulfate and Oyster Shell for Enhanced Bone Tissue Regeneration", 《ACS APPL. MATER. INTERFACES》 * |
| 寇冬权等: "牡蛎壳粉/α-半水硫酸钙复合人工骨修复兔股骨髁缺损的实验研究", 《医学研究杂志》 * |
| 张森林: "珊瑚与骨髓基质细胞和富血小板血浆构建组织工程骨的实验研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
| 张森林等: "富血小板血浆对珊瑚支架上骨髓基质细胞增殖和成骨分化的影响", 《口腔医学研究》 * |
| 张森林等: "富血小板血浆对珊瑚支架上骨髓基质细胞异位成骨的影响", 《口腔医学研究》 * |
| 张森林等: "应用珊瑚/骨髓基质细胞/富血小板血浆组织工程骨修复兔颅骨缺损", 《中国口腔颌面外科杂志》 * |
| 苏寒等: "应用兔骨髓基质细胞膜片与珊瑚支架构建组织工程骨", 《中国口腔领面外科杂志》 * |
| 薛恩兴等: "牡蛎壳材料填充兔股骨骨缺损的实验研究", 《浙江创伤外科》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106362213A (en) * | 2016-11-02 | 2017-02-01 | 中国人民解放军第三军医大学第三附属医院 | PRP loaded slow release antibiotic-mesenchymal stem cell gel system and preparation method thereof |
| CN106362213B (en) * | 2016-11-02 | 2019-11-19 | 中国人民解放军第三军医大学第三附属医院 | A kind of PRP load slow release antibiotic-mescenchymal stem cell gel systems and preparation method thereof |
| CN108187133A (en) * | 2018-01-09 | 2018-06-22 | 楼毅 | A kind of preparation method of medical composite type α-half-H 2 O calcium sulphate artificial bone |
| CN110694114A (en) * | 2019-10-31 | 2020-01-17 | 南通大学附属医院 | PRP-loaded pancreas acellular scaffold and preparation method thereof |
| CN112791236A (en) * | 2020-12-18 | 2021-05-14 | 广东普罗凯融生物医药科技有限公司 | Cell microcarrier matrix and application thereof |
| CN112717204A (en) * | 2021-01-19 | 2021-04-30 | 郭燕庆 | Autologous bone graft activity substitute composition, preparation method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lyons et al. | The healing of bony defects by cell-free collagen-based scaffolds compared to stem cell-seeded tissue engineered constructs | |
| CN105749348A (en) | Preparation method of PRP (platelet-rich plasma)/MSCs (marrow stromal cells)/oyster shell bone tissue scaffold material | |
| US6884621B2 (en) | Method and carrier for culturing multi-layer tissue in vitro | |
| Green et al. | Natural marine sponge fiber skeleton: a biomimetic scaffold for human osteoprogenitor cell attachment, growth, and differentiation | |
| CN101163512B (en) | Cartilaginiform and osteochondral sustitute comprising a multilayer structure | |
| CN105311681B (en) | Injectable composite material for bone repair and preparation method thereof | |
| CN1973910B (en) | Tissue engineering bone | |
| Kumbhar et al. | In vitro and in vivo studies of a novel bacterial cellulose-based acellular bilayer nanocomposite scaffold for the repair of osteochondral defects | |
| CN106456837A (en) | Methods for preparation of a terminally sterilized hydrogel derived from extracellular matrix | |
| Yu et al. | Autologous platelet-rich plasma induces bone formation of tissue-engineered bone with bone marrow mesenchymal stem cells on beta-tricalcium phosphate ceramics | |
| Weisgerber et al. | A mineralized collagen-polycaprolactone composite promotes healing of a porcine mandibular defect | |
| CN102271722A (en) | Cell-scaffold constructs | |
| ES2356678T3 (en) | BIOMATRIZ OF COLLAGEN AND METHOD TO PRODUCE. | |
| Chen et al. | A three-dimensional (3D) printed biomimetic hierarchical scaffold with a covalent modular release system for osteogenesis | |
| Viateau et al. | Comparative study of the osteogenic ability of four different ceramic constructs in an ectopic large animal model | |
| BRPI1010619B1 (en) | multidimensional biomaterial and method for its production | |
| Bagher et al. | Comparative study of bone repair using porous hydroxyapatite/β-tricalcium phosphate and xenograft scaffold in rabbits with tibia defect | |
| CN101417151A (en) | Biology source type calcified-layer containing cartilage tissue engineer bracket | |
| Zhao et al. | The study of the feasibility of segmental bone defect repair with tissue-engineered bone membrane: a qualitative observation | |
| CN101564555B (en) | Tissue engineering bone implant and method for constructing the same | |
| Ventura et al. | Bone regeneration using hydroxyapatite sponge scaffolds with in vivo deposited extracellular matrix | |
| CN1846793B (en) | Tissue engineering bone and its construction and application | |
| CN102971019B (en) | The method being engineered for complex organization | |
| Jagodzinski et al. | Bone marrow-derived cell concentrates have limited effects on osteochondral reconstructions in the mini pig | |
| Wang et al. | The effects of oyster shell/alpha-calcium sulfate hemihydrate/platelet-rich plasma/bone mesenchymal stem cells bioengineering scaffold on rat critical-sized calvarial defects |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160713 |