CN106075581A - A kind of method preparing self-bone grafting osteogenic formulation - Google Patents
A kind of method preparing self-bone grafting osteogenic formulation Download PDFInfo
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- CN106075581A CN106075581A CN201610420637.5A CN201610420637A CN106075581A CN 106075581 A CN106075581 A CN 106075581A CN 201610420637 A CN201610420637 A CN 201610420637A CN 106075581 A CN106075581 A CN 106075581A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
-
- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
- A61L27/3645—Connective tissue
- A61L27/365—Bones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—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 physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
-
- 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
-
- 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
Abstract
The present invention provides a kind of method preparing self-bone grafting osteogenic formulation, and the method comprises the steps: that 1. carry out decalcification process to 2 parts of teeth matrixes；Carry out the 2 parts of teeth matrixes processed through decalcification the most again extracting bone morphogenetic protein；The teeth matrix that the bone morphogenetic protein that relief extracts processes with 1 part of decalcification therein mixes, thus forms induced osteogenesis preparation.
Description
Technical field
The present invention relates to a kind of preparation repairing bone injury, particularly, particularly relate to a kind of induction osteogenesis, promote
Osseous tissue forms or repairs preparation and the method for bone injury.
Background technology
Self-bone grafting skeletonization technology is a kind of technology that current surgery reduction and periodontal reconstructions are conventional, mainly utilize self-bone grafting because of
Son activates bone lengthening and produces the function new bone of generation.Bone-inducing factor is more, and existing research focuses primarily upon bone morphogenetic protein
(bone morphogenetic protein.BMP) family protein research, bone morphogenetic protein belongs to low molecular sugar protide,
Have now been found bone morphogenetic protein l-14 multiple protein composition, wherein the bone-inducting active of bone morphogenetic protein 2 is the strongest,
Other bone morphogenetic protein composition has synergism to bone morphogenetic protein 2, common participation induced osteogenesis process.There is scholar
Think that bone morphogenetic protein is the one of TGF-1 3 (TGF 1) superfamily, it have obvious osteanagenesis and
Integrated implant ability, it is possible to induction cartilage and bone formation, and can be converted into key thin by the mesenchymal cell around induction of vascular
Born of the same parents, the bone morphogenetic protein that it is formed with extracellular matrix macromolecule interaction is the basis regulating bone formation effect.Bone
Morphogenetic proteins is widely present in bone matrix and the teeth matrix of mammal, is a kind of without species specificity, belongs to local
The acidoglycoprotein of somatomedin, but os in os morphogenetic proteins gradually decreases with the increase at age, and in teeth matrix
Bone morphogenetic protein has no minimizing.Also have research display decalcification teeth matrix (demineralized dental matrix,
DDM) in, the crude extract of bone morphogenetic protein is homogenous quantities decalcified bone matrix (demineralized bone matrix, DBM)
In 10 times, correspondingly decalcification teeth matrix induced osteogenesis amount in muscle should be 10 times of homogenous quantities decalcified bone matrix.But, single
Pure bone morphogenetic protein spreads too fast in vivo, the most easily by proteases for decomposing, thus can not act within the effective time
In more target cell, its induced activity is difficult to be fully played；The purification of bone morphogenetic protein is produced relatively difficult,
Relatively costly, it is difficult to be widely popularized use in clinic；Studies have found that the bone morphogenetic protein that purification is high can not obtain relatively
High biotic induce activity, is only used in combination with corresponding bone matrix, ability effectively induced osteogenesis, this is because collagen and substrate
May have impact on the calcium in hydroxyapatite crystal and phosphate ions, Organic substance is likely to play in a crystallization
The heart.Though having many reports about the study on the carrier of bone morphogenetic protein, as use hydroxyapatite, phosphate DFP, polyester,
The carriers such as collagen, demineralized bone matrix, titanium oxide, clot, but the combination between bone morphogenetic protein and carrier, composite square
Method, compositely proportional, do not have unified conclusion yet.Therefore, the direction of current research transfers the development of plyability bone induction material to.
Research thinks that decalcification teeth matrix (demineralized dentin matrix, DDM) is that one is sent out rich in Bones morphology
The complex of raw albumen, collagen and other proteoglycan, is the life of a kind of novel human homology's property with bone inductive effect
Thing active material, it, rich in multiple osteoinductive protein and the complex of carrier thereof, defines the sky of a kind of bone morphogenetic protein
So slowly discharge system.Such as Chinese invention patent 95112416.1；Or Chinese invention patent application, application number
201310602830.7；Application number 201310602878.8 describes and obtains these by dental treatments and can promote that bone is hindered
Mouth healing or the material of regeneration.It addition, also add some compositions in teeth matrix, and then the effect of improvement teeth matrix, such as
Description in Chinese invention patent application, application number 201510089391.3 and application number 201110129457.9.
Certainly, also have and use vivoexpression bmp protein then to mix with bone powder or other powder, this method system
Standby repair materials, actual effect is unsatisfactory, and mainly bmp protein the most easily loses activity, and the mixing of these powder
After, it is applied in the skeleton of damage, the most degraded by enzymes also loses activity.Therefore the activity of BMP is only kept in vivo,
Can preferably reach to promote the growth of osseous tissue.
Process as described above, although the reparation to bone impingement has certain effect, but yet suffers from a lot of solid
Some defects, be mainly reflected in following some: (1) tooth as activity BMP limited source, it is impossible to meet growing city
Need, depend teeth matrix after all alone as raw material, limit the development of industry；(2) after tooth, skeleton being carried out pulverization process,
Generally carry out decalcification at acid solution, it is thus achieved that containing the powder of activated protein BMP, although but soda acid etc. process can discharge calcium from
Son, exposes bmp protein, and simultaneously the most also albumen to BMP has damage greatly because BMP degeneration the most in acid condition and
Lose activity；This reduces the utilization ratio of bmp protein.
Therefore, it is necessary to current methods is improved, or new method is used to obtain bone renovating material.
Summary of the invention
On the one hand, the present invention provides a kind of method preparing self-bone grafting osteogenic formulation, and the method comprises the steps: that 1. is right
Teeth matrix carries out decalcification process；The most again to adding external source activated protein BMP, i.e. bone shape in the teeth matrix that decalcification processes
State generation albumen, thus form induced osteogenesis preparation.
On the other hand, the present invention provides a kind of method preparing self-bone grafting osteogenic formulation, and the method comprises the steps: 1.
2 parts of teeth matrixes are carried out decalcification process；The most again the 2 parts of teeth matrixes processed through decalcification are removed Bones morphology generation egg
White process；The teeth matrix that the bone morphogenetic protein that relief extracts processes with 1 part of decalcification therein mixes, thus shape
Become induced osteogenesis preparation.
On the other hand, the present invention provides a kind of method preparing self-bone grafting osteogenic formulation, and the method comprises the steps: 1.
External source bone matrix is carried out decalcification process；The bone matrix processed through decalcification is removed albumen the most again process；3. allow
External source bone morphogenetic protein mixes with the bone matrix processed through step 2, allows foreign protein form induction with bone matrix
Osteogenic formulation.
Some preferred embodiment in, a kind of method that decalcifying tooth tooth is carried out decalcification process includes walking as follows
Rapid: (1), mechanically dental tissue is ground into granule or lyophilized powder；(2), by dental tissue granule or lyophilized powder
It is placed in 0.65-0.72 equivalent hydrochloric acid solution to soak, soaks under 36 degrees Celsius and complete decalcification in 12 hours；(3), after decalcification, product is low
Temperature is dried and obtains freeze-dried powder.
Preferably, freeze-dried powder soaks 30 minutes in the hydrogen peroxide of 35 degree 3% Celsius and i.e. obtains decalcifying tooth matrix.
Preferably, described people's teeth matrix freeze-dried powder or granule, under conditions of Celsius temperature 5 DEG C, it is placed in 70% wine
Seminal fluid is stored standby.
Preferably, after decalcification product behaviour teeth matrix granule be placed in alcohol liquid store standby.Wherein, the concentration of ethanol is
Alcoholic solution under any concentration, such as 70%, 60%, 50%, 75%, 78%, 80%, 85% or 95%.
In preferred mode, first by tooth with 50% disinfectant solution (such as containing the sodium hypochlorite of effective ingredient 1.2%)
Soaking disinfection raw material tooth, the soaking disinfection time is 45 minutes, temperature is 36 degree Celsius；Mechanically remove on tooth body without using
The part being worth；Be ground into the dental tissue of granule or lyophilized powder with 1.2 sodium hypochlorite soaking disinfections, the soaking disinfection time is
45 minutes, temperature be 36 degree Celsius.Then in the pulverization process carrying out tooth.
Here tooth can be the tooth of people, it is possible to be the tooth of any mammal.It is preferably people's tooth.
In some preferred modes, to passing through the tooth substrate interpolation external source bmp protein that decalcification processes, outside interpolation
The method of source protein is as follows: first allow the teeth matrix powder of decalcification be suspended in the phosphate buffer of PH=6.0-7.5 (100
Gram 1 liter of buffer of configuration), then external source BMP, with the dissolving of the phosphate buffer of identical PH=6.0-7.5, (concentration is 1.0M
Every liter), then both mix, be put in whisking machines and carry out uniform speed slow stirring, the speed of stirring be 12-120 turn/
Minute, at room temperature it is stirred 12-24 hour simultaneously, stirring when, measured in mixed once solution every 3 hours
The concentration of bmp protein, the 20%-35% (0.2-0.35M/L) until lowering of concentration to initial BMP concentration stops stirring below,
Being then centrifuged for, remove supernatant fluid, remaining powder filters, and is then passed through cold drying and obtains people's teeth matrix, Celsius
Be placed under conditions of temperature 5 DEG C 70% alcohol liquid is stored standby.
In some preferred modes, 2 parts of people's teeth matrixes carrying out decalcification process, the substrate then processed decalcification is carried out
The extraction of albumen, then allows the bmp protein extracted carry out mixed processing with a people's teeth matrix therein.Processing method is: first
First allowing through decalcification, the teeth matrix powder that deproteinization (BMP activated protein and or other foreign proteins) extracts is suspended in PH=7.0
Phosphate buffer in (120 grams configuration 1 liter of buffer), then external source BMP (bmp protein extracted from 2 parts of substrate)
Dissolving with phosphate buffer (PH=7.0), then both mix, and are put in whisking machines and carry out uniform speed slow and stir
Mixing, the speed of stirring is 15-120 rev/min, is at room temperature stirred 12-24 hour simultaneously, stirring when, often
The concentration of the bmp protein of mixed once solution is measured, until the 20%-35% of lowering of concentration to initial BMP concentration every 3 hours
Hereinafter stopping stirring, be then centrifuged for, remove supernatant fluid, remaining powder filters, and then cold drying obtains people Ya Ji
Matter.Preferably, be placed in alcohol liquid under conditions of Celsius temperature 5 DEG C store standby.
In some preferred modes, to fresh external source skeleton (people's bone or the bone of mammal) (people here
Skeleton that bone mainly doctor takes off from patient body or the fresh skeleton of donation) carry out decalcification process,
The method that decalcification processes is as follows: skeletal tissue is mechanically ground into granule or lyophilized powder；(2), skeleton is made
Granule or lyophilized powder be placed in the hydrochloric acid solution of 0.65-0.72 equivalent immersion, under 36 degrees Celsius soak 24-48 hour
Complete decalcification；(3) after decalcification, product cold drying obtains bone powder or the granule of decalcification.Double with 35 degree Celsius 3% again
Oxygen water soaking i.e. obtains decalcification substrate in 30 minutes；Or, described substrate, under conditions of Celsius temperature 5 DEG C, it is placed in alcohol liquid
Middle storage is standby.Preferably, after decalcification product behaviour teeth matrix granule be placed in alcohol liquid store standby.Wherein, ethanol is dense
Degree is the alcoholic solution under any concentration, such as 70%, 60%, 50%, 75%, 78%, 80%, 85% or 95%.
Then the bone powder to decalcification carries out Deproteinated process, and Deproteinated processing procedure is as follows: the bone after decalcification
Bone powder adds suitable quantity of water, heat treated 56-72 hour in water-bath, and temperature is 35-40 degree Celsius, is then centrifuged, goes
Fall supernatant fluid, supernatant fluid is carried out the removal of the extraction of bmp protein, concentration and foreign protein.Or, bone powder adds pancreas
In enzyme or carase buffer solution, process more than 3-4 days under temperature is 37-40 degree Celsius, be then centrifuged supernatant
Liquid, thus by ferment treatment by albuminolysis, retain the precipitation after being centrifuged, thus Deproteinated bone powder is provided.
Preferably, allowing the bone powder and external source bmp protein mixed processing that decalcification and deproteinization process, the step of process is such as
Under: (120 grams are every first to allow the bone powder extracted through bmp protein after decalcification be suspended in the phosphate buffer of PH=7.0
Rising, then external source BMP phosphate buffer is dissolved (1.2M every liter), then both mix, and are put into whisking machines
On carry out uniform speed slow stirring, the speed of stirring is 30-80 rev/min, at room temperature or enters under 25-30 degree Celsius simultaneously
Row stirring 12-24 hour, stirring when, measured the concentration of the B MP albumen of mixed once solution every 3 hours, until dense
Degree drops to the 20%-25% (0.24-0.25M/L) of initial BMP concentration and stops stirring below, then by centrifugal, remove on
Clear liquid body, remaining powder filters, and is then passed through cold drying and obtains substrate.Preferably, in the condition of Celsius temperature 5 DEG C
Under be placed in 70% alcohol liquid is stored standby.
Preferably, external source bmp protein is BMP-2, BMP-4 and BMP-7 (Shanghai wheat bin Bioisystech Co., Ltd).Preferably
, external source bmp protein is BMP-2.Or, it is preferred that wherein, described external source bmp protein is BMP-2, BMP-1, BMP-
One or several in 3BMP-4, BMP-5, BMP-6 or BMP-7.The BMP activated protein of these commercializations can be by commercialization
Approach be commercially available.
Here powder or preparation are all the preparation of freeze-dried powder or other form.Here preparation and reagent, become
Divide or material is the meaning being equal to.
Beneficial effect
Bone tissue or the preparation of reparation and the effect phase of conventional people's teeth matrix is promoted through what the present invention provided
When, have is also better than existing people's teeth matrix, thus provides some products that can substitute, and reduces the cost of product, meets day
The market demand that benefit increases.
Accompanying drawing explanation
Fig. 1 is examples of implementation 1-3 and comparison CK affects comparative result figure to osteoblastic proliferation.
Fig. 2 is that examples of implementation 1,4,5 and comparison CK affect comparative result figure to osteoblastic proliferation.
Fig. 3 is examples of implementation 1-3 and comparison CK is to MC-3T3 cell ALP activity influence comparative test result figure.
Fig. 4 be examples of implementation Isosorbide-5-Nitrae and 5 with compare CK to MC-3T3 cell ALP activity influence comparative test result figure.
Detailed description of the invention
Examples of implementation 1: people's teeth matrix carries out decalcification and processes interpolation external source bmp protein.
1. to carry out the step of decalcification process as follows for people's teeth matrix:
(1), people's tooth of collection is placed in fresh water container, condition storage below 5 degrees Celsius；
(2), with 1.2% sodium hypochlorite soaking disinfection raw material tooth, the time is 55 minutes, temperature be 30 degrees Celsius (or its
Its problem, such as 35,30,38,40 degrees Celsius)；
(3) part having no value for use on tooth body, is removed；
(4), mechanically by dental tissue be ground into granule or lyophilized powder, mean diameter be 0.25 millimeter (such as
It can be 0.2,0.5,0.6,0.7,0.8,1.0,1.2 millimeters)；
(5), the dental tissue of granule or lyophilized powder, soaking disinfection it are ground into again with 1.2% sodium hypochlorite soaking disinfection
Time is 45 minutes, and temperature is 35-40 degree Celsius；
(6), dental tissue granule or lyophilized powder are placed in 0.65 (or 0.67,0.68,0.67,0.72) equivalent salt
Acid soak, soaks under 38 degrees Celsius and completes decalcification in 16 hours；
(7), cold drying, then the hydrogen peroxide dipping 30 minutes with 35 degree Celsius 3%.Through detection, take off through the method
The teeth matrix granule of Calcium treatment or lyophilized powder fine uniform, form is consistent, and in pole shape, length is about 100-200nm, directly
Footpath is about 50-lOOnm.
2, the interpolation of external source bmp protein.
A, step (7) is passed through decalcification teeth matrix process tooth substrate 1000 grams add external source bmp protein, add
The method of foreign protein is as follows:
B, the decalcification teeth matrix first step (7) processed remove hydrogen peroxide, rinse repeatedly until without dioxygen with aquesterilisa
Water exists；Obtain people's teeth matrix powder of 100 grams；
C, then the teeth matrix powder of decalcification is allowed to be suspended in the phosphate buffer of 1 liter of PH=7.2；
D, external source BMP-2 (1.0 grams, buy from the Shanghai wheat bin Bioisystech Co., Ltd) PH=of same volume
The phosphate buffer of 7.2 dissolves, and then both mix, and are put in whisking machines and carry out uniform speed slow stirring, stirring
Speed be 30-80 rev/min and be stirred, being simultaneously in the water-bath of alternating temperature, temperature is as follows: 26 degrees Celsius, and 8 is little
Time；30 degrees Celsius；6 hours；28 degrees Celsius；8 hours, 28 degrees Celsius, 12 hours；Then it is maintained under constant temperature 30 degrees Celsius and carries out
Continue stirring, measured the concentration of BMP-2 albumen in mixed once solution every 3 hours, until lowering of concentration is dense to initial BMP-2
Below the 20%-25% of degree stops stirring, then by centrifugal, removes supernatant fluid, and remaining powder filters, then
People's teeth matrix freeze-dried powder is obtained through cold drying.Through detection, through the method decalcification process teeth matrix granule or
Person's lyophilized powder fine uniform, form is consistent, and in pole shape, length is about 100 220nm, and diameter is about 40 1 11Onm.
Examples of implementation 2: people's teeth matrix carries out decalcification and processes interpolation external source bmp protein.
Being with examples of implementation 1 difference, foreign protein is BMP-7, other condition all as.
Examples of implementation 3: people's teeth matrix carries out decalcification and processes interpolation external source bmp protein.
Being with examples of implementation 1 difference, be simultaneously in the water-bath of constant temperature, temperature is 15,20,35,38 Celsius
Degree, the time was more than 34 hours, measured the concentration of BMP-2 albumen in mixed once solution every 3 hours, until lowering of concentration arrives
Below the 20%-25% of initial BMP-2 concentration stops stirring, then by centrifugal, removes supernatant fluid, and remaining powder enters
Row filters, and is then passed through cold drying and obtains people's teeth matrix, is placed in 70% alcohol liquid storage under conditions of Celsius temperature 5 DEG C
Standby.Through detection, through teeth matrix granule or the lyophilized powder fine uniform of the method decalcification process, form is consistent, in
Pole shape, length is about 100 220nm, and diameter is about 40 1 11Onm.
Examples of implementation 4:2 part people's teeth matrix carries out decalcification and deproteinization (BMP) processes, and processes the BMP egg albumen extracted The people's teeth matrix powder mixing processed through decalcification deproteinization with portion。
1. to carry out the step of decalcification process as follows for people's teeth matrix:
(1), people's tooth of collection is placed in fresh water container, condition storage below 5 degrees Celsius；
(2), with 1.2% sodium hypochlorite soaking disinfection raw material tooth, the time is 55 minutes, temperature be 30 degree Celsius (or its
Its problem, such as 35,30,38,40 degrees Celsius)；
(3) part having no value for use on tooth body, is removed；
(4), mechanically by dental tissue be ground into granule or lyophilized powder, mean diameter be 0.25 millimeter (such as
It can be 0.2,0.5,0.6,0.7,0.8,1.0,1.2 millimeters)；
(5), the dental tissue of granule or lyophilized powder, soaking disinfection it are ground into again with 1.0% sodium hypochlorite soaking disinfection
Time is 45 minutes, and temperature is 35-40 degree Celsius；
(6), dental tissue granule or lyophilized powder are placed in 0.65 (or 0.67,0.68,0.67,0.72) equivalent salt
Acid soak, soaks under 38 degrees Celsius and completes decalcification in 20 hours；
(7), cold drying, then the hydrogen peroxide dipping 30 minutes with 35 degree Celsius 3%.
2. carry out the extraction process of bmp protein.
1000 grams of powder of tooth substrate that step (7) passes through the process of decalcification teeth matrix carry out extracting the place of albumen
Reason, the method for the albumen extracting BMP is as follows:
A, the decalcification teeth matrix first step (7) processed remove hydrogen peroxide, rinse repeatedly until without dioxygen with aquesterilisa
Water exists；Obtain people's teeth matrix powder of 1000 grams；
B, then 1000 grams of the teeth matrix powder of decalcification is allowed to be suspended in the phosphate buffer of 5 liters of PH=7.2；
C, in water-bath the phosphate buffer of heat treated b step 280 hours, temperature is 40 degrees Celsius, heating
During process, constantly stir；Then it is centrifuged, separates supernatant and deposit, supernatant molecule is sieved
From purification and the concentration of destination protein, final acquisition bmp protein activity extract, through measuring, its molecular weight is the molecule of BMP
Amount is 20--50kda.Precipitation powder is filtered, is dried, reclaim, it is thus achieved that 980 grams of teeth matrix powder processed through deproteinization
End.
D, allowing teeth matrix powder that decalcification and deproteinization the process bmp protein mixed processing with extraction, the step of process is such as
Under: first allow the teeth matrix powder extracted through bmp protein after decalcification be suspended in the phosphate buffer of PH=7.0 (100 grams
Every liter, configure 5 liters of solution, add up to 500 grams) then the bmp protein (step c) phosphoric acid extracted from 1000 grams of people's teeth matrixes
Salt buffer dissolve, then both mix, be put in whisking machines and carry out uniform speed slow stirring, the speed of stirring for for
30-80 rev/min, at room temperature or it is stirred 12-24 hour under 25-30 degree Celsius simultaneously, stirring when, every
The concentration of bmp protein measuring mixed once solution in 3 hours, until lowering of concentration to initial BMP concentration 10%-15% with
Lower stopping stirs, and then by centrifugal, removes supernatant fluid, and remaining powder filters, and is then passed through cold drying and obtains
Substrate, be placed under conditions of Celsius temperature 5 DEG C 70% alcohol liquid is stored standby.
Through detection, through teeth matrix granule or the lyophilized powder fine uniform of the method decalcification process, form is consistent,
In pole shape, length is about 80 180nm, and diameter is about 30 1 7Onm.
Examples of implementation 5: external source people's bone carries out decalcification, deproteinization processes, then add external source bmp protein.
1. to carry out the step of decalcification process as follows for people's bone:
(1), people's bone of collection is put into fresh water container, condition storage below 5 degrees Celsius；
(2), with 2.0% sodium hypochlorite soaking disinfection raw material skeleton, the time is 55 minutes, temperature be 37 degrees Celsius (or
Other temperature, such as 35,30,38,40 degrees Celsius)；
(3) part having no value for use on skeleton, is removed；
(4), mechanically by skeletal tissue being ground into granule, mean diameter is 0.25 millimeter and (can be such as
0.2,0.5,0.6,0.7,0.8,1.0,1.2 millimeters, or the nano-particle of other particle diameters, such as 50-100,120-200 nanometer
Granule)；
(5), again with 1.5% sodium hypochlorite soaking disinfection being ground into granule, the soaking disinfection time is 45 minutes, temperature is
35-40 degree Celsius；
(6), by skeletal tissue's granule or be placed in 0.65 (or 0.67,0.68,0.67,0.72) equivalent hydrochloric acid solution in
Soak, soak under 38 degrees Celsius and complete decalcification in 16 hours；
(7), cold drying obtain freeze-dried powder or granule, then the hydrogen peroxide dipping 30 minutes with 35 degree Celsius 3%.
Skeleton matter granule after detection, the method decalcification process or lyophilized powder fine uniform, form is consistent, in
Pole shape, length is about 120 240nm, and diameter is about 60 1 120nm.
2. carry out the degradation treatment of bmp protein.
Process side to 1000 grams of powder extraction albumen of the bone matrix passed through in step (7) after decalcification skeleton processes
Method is as follows:
A, first removing step (7) decalcification processes the hydrogen peroxide in bone matrix, rinses repeatedly until unparalleled with aquesterilisa
Oxygen water exists；Obtain the bone matrix powder of 1000 grams；Then the bone powder allowing 1000 grams of decalcifications is suspended in 2 liters of PH=7.2
Phosphate buffer in；
B, for making enzyme digestion reaction reach preferable degree, and combine Productive statistics cost, select pancreatin, enzymolysis in an experiment
Kettle temperature is adjusted to 48-50 DEG C, and pH value is adjusted to when 8.5 add pancreatin O.35g；The response time of enzyme is 12-56 hour, carries out
Protein degradation processes；After process terminates, it is centrifuged separating supernatant and precipitation, removes supernatant, retain precipitation, and do
Dry, it is thus achieved that 900 grams of Deproteinated bone powder of decalcification.
C, then the Deproteinated bone powder of decalcification 1000 grams is allowed to be suspended in the phosphate buffer of 5 liters of PH=7.2；
D, external source BMP-2 (1.0 grams, buy from the Shanghai wheat bin Bioisystech Co., Ltd) PH=of same volume
The phosphate buffer of 7.2 dissolves, and then both mix, and are put in whisking machines and carry out uniform speed slow stirring, stirring
Speed be 30-80 rev/min and be stirred, being simultaneously in the water-bath of alternating temperature, temperature is as follows: 26 degrees Celsius, and 8 is little
Time；30 degrees Celsius；6 hours；28 degrees Celsius；8 hours, 28 degrees Celsius, 12 hours；Then it is maintained under constant temperature 30 degrees Celsius and carries out
Continue stirring, measured the concentration of BMP-2 albumen in mixed once solution every 3 hours, until lowering of concentration is dense to initial BMP-2
Below the 5%-6% of degree stops stirring, is then centrifuged for, removes supernatant fluid, and remaining powder filters, and is then passed through low
Temperature is dried to obtain bone matrix, be placed under conditions of Celsius temperature 5 DEG C 70% alcohol liquid is stored standby.
Through detection, through bone matrix granule or lyophilized powder fine uniform, the form one of the method decalcification process
Causing, in pole shape, length is about 122 240nm, and diameter is about 60 1 110nm.
Examples of implementation 6: the compliance test result of self-bone grafting osteogenic formulation
1. material and method
1.1 experimental apparatus
ELISA Plate (24 holes, 96 holes), microplate reader (Model 550), electric centrifuge (80-2 type), cell culture incubator
(Heraeus BB6220 type), bidirectional magnetic force heating stirrer (Medical Instruments factory of Jintan City of Jiangsu Province), (Shanghai is accurate for acidometer
Scientific instrument company limited), thermostat water bath (Medical Instruments factory of Jintan City of Jiangsu Province), (Suzhou purifies medical superclean bench
Instrument factory.
1.2 experiment material
Derive from the material of the sub-1-5 of embodiments of the invention and Shenzhen light Chuan Bo biological product Development Co., Ltd
Commercial teeth matrix carries out parallel control (CK) and processes experiment, class standard hyclone (Hangzhou Ilex purpurea Hassk.[I.chinensis Sims), DMEM culture medium
(Sigma), 0.25% trypsin Sigma), Tritonx-100 (the raw work in Shanghai), ALP standard preparation box (the raw work in Shanghai).
2 experimental techniques
2.1 MC-3T3 osteoblasts cultivation:
MC-3T3 osteoblast routine is recovered in the culture medium containing 10% hyclone, 50ml/L CO2, saturated humidity,
Cultivate under 37 DEG C of environment, change liquid after 24h, after growing to 80%, after 2.5/L trypsinization, turn generation, do for cell with 3-4
Experiment
2.2 MC-3T3 osteoblastic proliferation researchs
By limited to substrate powder and the rich biological product development of Shenzhen's light wound of sub-for embodiments of the invention 1-5 before experiment
The commercial teeth matrix powder of company weighs 0.1 gram and adds 24 orifice plates, every kind of sample average 3 hole, and ultraviolet irradiates sterilizing in 30 minutes.
The cell dissociation of exponential phase being made cell suspending liquid, adjusts concentration, making inoculum density is 1 × 104/ ml, adds 24 holes
Plate, every hole 1ml, at 50ml/L CO2: cultivate 2 under saturated humidity, 37 DEG C of environment, 4, after 6d, select 4 successively in each culture plate
Hole, adds MTT liquid 160ul (microlitre), continues to cultivate 4h, discards culture fluid, adds DMSO1.2ml, and vibrate 10min, by 200 μ l
(microlitre) is placed in 96 orifice plates, average three holes of every sample, measures absorbance A value with enzyme-linked immunosorbent assay instrument at 490nm wavelength, uses
Mtt assay cell counting standard curve estimation cell density.By single dependent variable multifactor analysis of variance pair of SPSS statistical software
Above experimental result is analyzed.
Result is as follows:
The MTT test result of different disposal is shown in Fig. 1-2.Along with the prolongation of incubation time, each group cell quantity has increased
Add.The material inoculation group cell proliferating number of embodiments of the present invention 1,3,4,5 is apparently higher than matched group CK, through statistics credit
Analysis, the material of embodiment 1,3,4,5 with compare statistically significant (P < O.05) (Fig. 1 and Fig. 2) between CK.Further
Illustrate that the present invention obtains become bone matrix compared to existing technology in commercial substrate there is higher activity.There is application greatly
Prospect.
2.3 MC-3T3 cell ALP determination of activity
Cell inoculation method ibid, is cultivated 2,4, after 6d, is discarded culture fluid, and PBS (0.01mol/L) rinses 3 times, adds
2ml/L Triton X 800 μ l ((microlitre)), 4 DEG C of refrigerator overnight, add 300 microlitre ALP substrates, 37 DEG C keep 40min,
With 0.1mol/L KOH stopped reaction, by 200 μ l, ((microlitre) moves into 96 orifice plates, and average three holes of every sample, with enzyme-linked immunosorbent assay instrument
Measure absorbance A value in 410 nanometers, i.e. represent Cellular alkaline phosphatase activity.Different to alkaline phosphatase for getting rid of the rate of increase
The impact of enzymatic activity, revises as follows: osteoblast mean alkaline phosphatase activity=record absorbance A value/same period skeletonization
Cell proliferation rate.By single dependent variable multifactor analysis of variance of SPSS statistical software, above experimental result is analyzed.
Result is as follows:
See Fig. 3,4.From figure 3, it can be seen that embodiments of the invention 1 and 3 obtain teeth matrix activity apparently higher than
The activity of teeth matrix in prior art.It addition, also further demonstrate that BMP-2 albumen is main activated protein, for skeleton
Repair or increase and there is important effect.Extending with incubation time, each group Cellular alkaline phosphatase activity all increased, and implements
The alkaline phosphatase activities of example 1 and 3 is higher than matched group, through statistical analysis, examples of implementation 1 with compare CK, examples of implementation 3,4
And 5 respectively with compare between CK two groups statistically significant (P < 0.05), in significant difference.So further illustrate the present invention
Obtain become bone matrix compared to existing technology in commercial substrate there is higher activity.
It addition, prove (method of employing such as examples of implementation 1 to be added foreign protein and enforcement by our other experiment
The foreign protein added in example 5), in all external source bmp proteins, such as, external source bmp protein is BMP-2, BMP-1, BMP-
3, one or several in BMP-4, BMP-5, BMP-6 or BMP-7, the when of individually using a kind of foreign protein, external source egg
The effect of white BMP-2, BMP-3 and BMP-7 is better than other albumen.
It addition, matrix granule that examples of implementation 1,3,4,5 are obtained by we or dry powder and the rich biology of Shenzhen's light wound
The commercial teeth matrix of goods Development Co., Ltd carries out parallel control (CK) and processes experiment, investigates for senile fracture, secondary
Fracture and three phases, the repairing effect of fourth phase femur head necrosis bone.It was found that relative to the teeth matrix of existing commercial distribution
Material (becomes osseous granules or preparation), and matrix granule or the curative effect of dry powder that embodiments of the invention 1,3,4,5 obtains are bright
The curative effect of the aobvious material being better than the teeth matrix that the most commercial teeth matrix material, particularly examples of implementation 1,3,4 obtain is best, special
It not that examples of implementation 1 and 4 effect is more preferable.This is possibly due to, and existing teeth matrix material is directly over decalcification and processes, wherein
The activated protein contained is the most fewer, and in acid decalcification processing procedure, may cause activity bmp protein in a large number
Degenerative lesion, thus effect is less desirable, and decalcification processes and has bigger randomness, and the performance of product has the biggest
Differences between batches.And the present invention is processed by simple, curative effect can be significantly improved；It addition, from January ,-2016 in January, 2014
Preserving at normal temperatures, the result carrying out activity experiment shows, lower about 2 years of preservation under room temperature, although activity the most somewhat reduces (big
About reduce about 5-10%) (specific experiment process and concrete data are slightly), but remain in that higher activity, store 2 years
After, activity almost current commercial product activity 2-4 times.This commercially produces for being standardized in a large number, and effect is the most notable
Induced osteogenesis product provide a new approach.
Claims (6)
1. the method preparing self-bone grafting osteogenic formulation, the method comprises the steps: that 2 parts of teeth matrixes are carried out at decalcification by 1.
Reason；Carry out the 2 parts of teeth matrixes processed through decalcification the most again extracting bone morphogenetic protein；The bone shape that relief extracts
The teeth matrix that state generation albumen processes with 1 part of decalcification therein mixes, thus forms induced osteogenesis preparation.
Method the most according to claim 1, wherein, the method extracting bone morphogenetic protein is as follows: allow the tooth base of decalcification
Matter powder is suspended in the phosphate buffer of PH=7.2；Heat treated phosphate buffer 280 hours in water-bath, temperature
Degree, for 35-40 degree Celsius, during heat treated, constantly stirs；Then it is centrifuged, separates supernatant and precipitate
Matter, carries out purification and the concentration of conventional molecular sieve separation destination protein to supernatant, final acquisition bmp protein activity extract.
Method the most according to claim 1 and 2, wherein, the molecular weight of the active bmp protein of extraction is 20--50kda.
Method the most according to claim 3, wherein, by decalcification and carries the BMP small molecular protein and 1 part extracting acquisition
Take the teeth matrix granule after albumen or lyophilized powder carries out mixed processing, allow the little molecule of BMP and granule or lyophilized powder enter
Row combines.
Method the most according to claim 4, wherein, the method that described combination processes is as follows: first allow decalcification, through egg
In the white 2 parts of teeth matrix powder extracted 1 part is suspended in the phosphate buffer of PH=7.0, then from 2 parts of teeth matrixes
The bmp protein extracted phosphate buffer dissolves, and then both mix, and are put in whisking machines and carry out uniform speed slow
Stirring, the speed of stirring is 20-120 rev/min, at room temperature or is stirred 12-56 under 25-30 degree Celsius little simultaneously
Time, stirring when, measured the concentration of the bmp protein of mixed once solution every 3 hours, until lowering of concentration is to initial
Below the 10%-15% of BMP concentration stops stirring, then by centrifugal, removes supernatant fluid, and remaining powder filters,
It is then passed through cold drying.
6. become bone granule or a freeze-dried powder preparation by the self-bone grafting of one of claim 1-5 preparation, wherein said
The granule of preparation or lyophilized powder are pole shape, and length is about 80 180nm, and diameter is about 30 1 70nm.
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| CN201610420637.5A CN106075581A (en) | 2016-06-13 | 2016-06-13 | A kind of method preparing self-bone grafting osteogenic formulation |
| CN201610904981.1A CN107496986A (en) | 2016-06-13 | 2016-10-17 | A kind of method for preparing self-bone grafting osteogenic formulation |
| EP17175408.8A EP3257522A1 (en) | 2016-06-13 | 2017-06-12 | Method for preparing an induced osteogenesis formulation |
| JP2017115519A JP6381743B2 (en) | 2016-06-13 | 2017-06-12 | Method for preparing osteoinductive osteogenic preparation |
| US15/619,705 US10576159B2 (en) | 2016-06-13 | 2017-06-12 | Method for preparing an induced osteogenesis formulation |
| KR1020170073760A KR20170140782A (en) | 2016-06-13 | 2017-06-13 | Method for preparing an induced osteogenesis formulation |
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| CN103990180A (en) * | 2014-05-26 | 2014-08-20 | 中国人民解放军第四军医大学 | Preparation method and application of deproteinated decalcification bone matrix implantable microcarrier |
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