CN107496987A - A kind of method for preparing self-bone grafting osteogenic formulation - Google Patents
A kind of method for preparing self-bone grafting osteogenic formulation Download PDFInfo
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- CN107496987A CN107496987A CN201610906514.2A CN201610906514A CN107496987A CN 107496987 A CN107496987 A CN 107496987A CN 201610906514 A CN201610906514 A CN 201610906514A CN 107496987 A CN107496987 A CN 107496987A
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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
- A61L27/3604—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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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
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- 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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- 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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- 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
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- 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
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- 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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- 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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- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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Abstract
The present invention provides a kind of method for preparing self-bone grafting osteogenic formulation, and this method comprises the following steps：1. pair external source bone matrix carries out decalcification processing；2. and then albumen is removed to the bone matrix by decalcification processing again；3. allow external source bone morphogenetic protein and the bone matrix that is handled by step 2 to be mixed, foreign protein and estimate shape induced osteogenesis preparation are allowed.
Description
Technical field
The present invention relates to a kind of preparation for repairing bone injury, and particularly, more particularly to one kind one kind induces ostosis, promotes
Bone tissue forms or repaired the preparation and method of bone injury.
Background technology
Self-bone grafting skeletonization technology is the conventional a kind of technology of current surgery reduction and periodontal reconstructions, mainly using self-bone grafting because
Son activation bone lengthening and production function produce new bone.Bone-inducing factor is more, and now research focuses primarily upon bone morphogenetic protein
(bone morphogenetic protein.BMP) family protein is studied, bone morphogenetic protein category low molecular sugar protide,
It has now been found that bone morphogenetic protein l-14 multiple protein compositions, the bone-inducting active of wherein bone morphogenetic protein 2 is most strong,
Other bone morphogenetic protein compositions have synergy to bone morphogenetic protein 2, common to participate in induced osteogenesis process.There is scholar
It is one kind of TGF-1 3 (TGF 1) superfamily to think bone morphogenetic protein, it have obvious osteanagenesis and
Integrated implant ability, can induce cartilage and bon e formation, and can be around induction of vascular mesenchymal cell be converted into it is key thin
Born of the same parents, the bone morphogenetic protein that it is formed with extracellular matrix macromolecule interaction are the bases for adjusting bon e formation effect.Bone
Morphogenetic proteins are widely present in the bone matrix and teeth matrix of mammal, are a kind of no species specificities, and category is local
The acidoglycoprotein of growth factor, but os in os morphogenetic proteins gradually decrease with the increase at age, and in teeth matrix
Bone morphogenetic protein has no reduction.Also research show decalcification teeth matrix (demineralized dental matrix,
DDM the crude extract of bone morphogenetic protein is homogenous quantities decalcified bone matrix (demineralized bone matrix, DBM) in)
In 10 times, correspondingly decalcification teeth matrix induced osteogenesis amount in muscle should be 10 times of homogenous quantities decalcified bone matrix.It is but single
Pure bone morphogenetic protein spreads too soon in vivo, also easily by proteases for decomposing, thus can not be acted within the effective time
In more target cells, its induced activity is difficult to be fully played；The purifying of bone morphogenetic protein produce it is relatively difficult,
Cost is higher, it is difficult to is widely popularized use in clinic；Studies have found that the high bone morphogenetic protein of purifying can not obtain compared with
High biological induced activity, is only used in combination with corresponding bone matrix, the effective induced osteogenesis of ability, because collagen and matrix
The calcium and phosphate ions in hydroxyapatite crystal may be have impact on, organic matter may also be played in a crystallization
The heart.Though the study on the carrier on bone morphogenetic protein has many reports, such as using hydroxyapatite, phosphate DFP, polyester,
The carriers such as collagen, demineralized bone matrix, titanium oxide, clot, but combination, composite square between bone morphogenetic protein and carrier
Method, compositely proportional, there is not unified conclusion yet.Therefore, the direction of current research switchs to the development of plyability bone induction material.
Research thinks that decalcification teeth matrix (demineralized dentin matrix, DDM) is that a kind of Bones morphology that is rich in is sent out
The compound of raw albumen, collagen and other proteoglycans, it is a kind of life of new human homology's property with bone inductive effect
Thing active material, its compound rich in a variety of osteoinductive proteins and its carrier, forms a kind of day of bone morphogenetic protein
So slowly discharge system.Such as Chinese invention patent 95112416.1；Or Chinese invention patent application, application number
201310602830.7；Described in application number 201310602878.8 and obtain these by handling tooth and bone can be promoted to hinder
Mouth healing or the material of regeneration.In addition, also adding some compositions in teeth matrix, and then the effect of teeth matrix is improved, such as
Chinese invention patent application, the description in application number 201510089391.3 and application number 201110129457.9.
Certainly, also using vivoexpression bmp protein and then and bone powder or other powder mixing, this method system
Standby repair materials, actual effect is unsatisfactory, and mainly bmp protein easily loses activity in vitro, and the mixing of these powder
Afterwards, it is applied in the bone of damage, is easily degraded by enzymes and also loses activity.Therefore BMP activity is only kept in vivo,
The growth for promoting bone tissue can preferably be reached.
Process as described above, although the reparation to bone impingement has certain effect, there are still many solid
The defects of having, be mainly reflected in it is following some：(1) limited source of the tooth as active BMP, it is impossible to meet growing city
Field needs, and depends teeth matrix alone after all as raw material, limits the development of industry；(2) after carrying out pulverization process to tooth, bone,
Generally acid solution carry out decalcification, obtain the powder containing activated protein BMP, although soda acid etc. processing can discharge calcium from
Son, exposure bmp protein, while also there is greatly damage to BMP albumen, because BMP is easily denatured in acid condition
Lose activity；This reduces the utilization ratio of bmp protein.
Therefore, it is necessary to be improved to current methods, or bone renovating material is obtained using new method.
The content of the invention
On the one hand, the present invention provides a kind of method for preparing self-bone grafting osteogenic formulation, and this method comprises the following steps：1. pair
Teeth matrix carries out decalcification processing；2. and then external source activated protein BMP, i.e. bone shape are added into the teeth matrix by decalcification processing again
Albumen occurs for state, so as to form induced osteogenesis preparation.
On the other hand, the present invention provides a kind of method for preparing self-bone grafting osteogenic formulation, and this method comprises the following steps：1.
Decalcification processing is carried out to 2 parts of teeth matrixes；2. and then Bones morphology is removed to 2 parts of teeth matrixes by decalcification processing again egg occurs
White processing；3. the bone morphogenetic protein of most relief extraction is mixed with the teeth matrix that 1 part of decalcification therein is handled, so as to shape
Into induced osteogenesis preparation.
On the other hand, the present invention provides a kind of method for preparing self-bone grafting osteogenic formulation, and this method comprises the following steps：1.
Decalcification processing is carried out to external source bone matrix；2. and then albumen processing is removed to the bone matrix by decalcification processing again；3. allow
External source bone morphogenetic protein is mixed with the bone matrix handled by step 2, is allowed foreign protein to be formed with bone matrix and is induced
Osteogenic formulation.
In some preferred embodiments, the method for decalcification processing is carried out to a kind of decalcifying tooth tooth includes following step
Suddenly：(1) dental tissue mechanically, is ground into granule or freeze-dried powder；(2), by dental tissue granule or freeze-dried powder
The immersion of 0.65-0.72 equivalents hydrochloric acid solution is placed in, is soaked under 36 degrees Celsius 12 hours and completes decalcification；(3), product is low after decalcification
Temperature, which is dried, obtains freeze-dried powder.
Preferably, freeze-dried powder soaks 30 minutes in 35 degree 3% Celsius of hydrogen peroxide obtains decalcifying tooth matrix.
Preferably, described people's teeth matrix freeze-dried powder or particle, 70% wine is placed under conditions of 5 DEG C of Celsius temperature
Stored in seminal fluid standby.
Preferably, after decalcification product behaviour teeth matrix particle be placed in alcohol liquid store it is standby.Wherein, the concentration of alcohol is
Alcoholic solution under any concentration, such as 70%, 60%, 50%, 75%, 78%, 80%, 85% or 95%.
In preferable mode, first by tooth with 50% thimerosal (such as sodium hypochlorite containing active 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 use
The part of value；The dental tissue of granule or freeze-dried powder is ground into 1.2 sodium hypochlorite soaking disinfections, the soaking disinfection time is
45 minutes, temperature be 36 degree Celsius.Then the pulverization process of tooth is being carried out.
Here tooth can be the tooth of people or the tooth of any mammal.Preferable is people's tooth.
In some preferable modes, external source bmp protein is added to the tooth substrate for having already passed through decalcification processing, addition is outer
The method of source protein is as follows：The teeth matrix powder of decalcification is allowed to be suspended in PH=6.0-7.5 phosphate buffer (100 first
Gram 1 liter of buffer solution of configuration), then external source BMP with identical PH=6.0-7.5 phosphate buffer dissolving (concentration 1.0M
Every liter), then both mix, and are put into whisking machines progress uniform speed slow stirring, and the speed of stirring turns for 12-120/
Minute, while 12-24 hours are stirred at room temperature, when stirring, every in 3 hours measurement mixed once solution
The concentration of bmp protein, the 20%-35% (0.2-0.35M/L) for dropping to initial BMP concentration until concentration stop stirring below,
It is then centrifuged for, removes supernatant fluid, remaining powder is filtered, and then people's teeth matrix is obtained by low temperature drying, Celsius
Be placed under conditions of 5 DEG C of temperature in 70% alcohol liquid store it is standby.
In some preferable modes, decalcification processing is carried out to 2 parts of people's teeth matrixes, then the matrix of decalcification processing carried out
The extraction of albumen, the bmp protein of extraction and a people's teeth matrix therein is then allowed to carry out mixed processing.Processing method is：It is first
First allow by decalcification, the teeth matrix powder of de- albumen (BMP activated proteins and or other foreign proteins) extraction is suspended in PH=7.0
Phosphate buffer in (120 grams configuration 1 liter of buffer solution), then external source BMP (bmp protein extracted from 2 parts of matrix)
Dissolved with phosphate buffer (PH=7.0), then both mix, and are put into progress uniform speed slow in whisking machines and stir
Mix, the speed of stirring is 15-120 revs/min, while is stirred 12-24 hours at room temperature, when stirring, often
Every the concentration of the bmp protein of 3 hours measurement mixed once solution, until concentration drops to the 20%-35% of initial BMP concentration
Stop stirring below, be then centrifuged for, remove supernatant fluid, remaining powder is filtered, and then low temperature drying obtains people Ya Ji
Matter.Preferably, it is placed under conditions of 5 DEG C of Celsius temperature in alcohol liquid and stores standby
In some preferable modes, to fresh external source bone (people's bone or the bone of mammal) (people here
Bone is mainly the skeleton that doctor removes from patient body or the fresh skeleton of donation) decalcification processing is carried out,
The method of decalcification processing is as follows：Skeletal tissue is mechanically ground into granule or freeze-dried powder；(2), bone is made
Granule or freeze-dried powder be placed in the hydrochloric acid solution of 0.65-0.72 equivalents and soak, under 36 degrees Celsius soak 24-48 hours
Complete decalcification；(3) product low temperature drying obtains the bone powder or particle of decalcification after decalcification.Again with 35 degree Celsius 3% of pair
Oxygen water, which soaks 30 minutes, obtains decalcification matrix；Or described matrix, it is placed in alcohol liquid under conditions of 5 DEG C of Celsius temperature
Middle storage is standby.Preferably, after decalcification product behaviour teeth matrix particle be placed in alcohol liquid store it is standby.Wherein, alcohol is dense
Spend for the alcoholic solution under any concentration, such as 70%, 60%, 50%, 75%, 78%, 80%, 85% or 95%.
Then Deproteinated processing is carried out to the bone powder of decalcification, Deproteinated processing procedure is as follows：Bone after decalcification
Bone powder adds suitable quantity of water, heats in water-bath 56-72 hours, and temperature is 35-40 degrees Celsius, is then centrifuged, gone
Fall supernatant fluid, extraction, concentration and the removal of foreign protein of bmp protein are carried out to supernatant fluid.Or bone powder adds pancreas
In enzyme or pawpaw enzyme buffer solution, handled more than 3-4 days under being 37-40 degrees Celsius in temperature, then carry out centrifugation and remove supernatant
Liquid, so as to, by protein breakdown, retain the precipitation after centrifugation, so as to provide Deproteinated bone powder by ferment treatment.
Preferably, the bone powder that decalcification and de- albumen are handled and external source bmp protein mixed processing are allowed, the step of processing such as
Under：Allowing the bone powder after decalcification by bmp protein extraction to be suspended in PH=7.0 phosphate buffer first, (120 grams every
Rise, external source BMP is then dissolved (every liter of 1.2M) with phosphate buffer, then both mix, and are put into whisking machines
Upper progress uniform speed slow stirring, the speed of stirring is 30-80 revs/min, while is entered at room temperature or under 25-30 degrees Celsius
Row stirring 12-24 hours, when stirring, every the concentration of the bmp protein of 3 hours measurement mixed once solution, Zhi Daonong
Degree drop to initial BMP concentration 20%-25% (0.24-0.25M/L) stops below stirring, then by centrifuge, remove on
Clear liquid body, remaining powder are filtered, and then obtain matrix by low temperature drying.Preferably, in the condition of 5 DEG C of Celsius temperature
Under be placed in 70% alcohol liquid store it is standby.
Preferably, external source bmp protein is BMP-2, BMP-4 and BMP-7 (Shanghai wheat bin Bioisystech Co., Ltd).It is preferred that
, 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 kinds in 3BMP-4, BMP-5, BMP-6 or BMP-7.These commercial BMP activated proteins can pass through commercialization
Approach be commercially available.
Here either preparation is all freeze-dried powder or the preparation of other forms to powder.Here preparation with reagent, into
Divide or material is the equivalent meaning.
Beneficial effect
By promotion bone tissue provided by the invention or the preparation of reparation and the effect phase of conventional people's teeth matrix
When some is also better than existing people's teeth matrix, so as to provide the product that some can be substituted, reduces the cost of product, meets day
The market that benefit increases needs.
Brief description of the drawings
Fig. 1 is examples of implementation 1-3 and compares influence comparative result figures of the CK to osteoblastic proliferation.
Fig. 2 is examples of implementation 1,4,5 and compares influence comparative result figures of the CK to osteoblastic proliferation.
Fig. 3 is examples of implementation 1-3 and control CK is to MC-3T3 cell ALP activity influence comparative test result figures.
Fig. 4 is examples of implementation Isosorbide-5-Nitrae and 5 with compareing CK to MC-3T3 cell ALP activity influence comparative test result figures.
Embodiment
Examples of implementation 1：People's teeth matrix carries out decalcification processing addition external source bmp protein.
It is as follows that 1. people's teeth matrix carries out the step of decalcification processing：
(1), people's tooth of collection is placed in fresh water container, the 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, for example, 35,30,38,40 degrees Celsius)；
(3) part being had no value for use on tooth body, is removed；
(4) dental tissue mechanically, is ground into granule or freeze-dried powder, average grain diameter be 0.25 millimeter (such as
It can be 0.2,0.5,0.6,0.7,0.8,1.0,1.2 millimeter)；
(5) dental tissue of granule or freeze-dried powder, soaking disinfection, are ground into 1.2% sodium hypochlorite soaking disinfection again
Time is 45 minutes, and temperature is 35-40 degree Celsius；
(6), by dental tissue granule, either freeze-dried powder is placed in 0.65 (or 0.67,0.68,0.67,0.72) equivalent salt
Acid soak, soaked under 38 degrees Celsius 16 hours and complete decalcification；
(7), low temperature drying, then with 35 degree Celsius 3% of hydrogen peroxide dipping 30 minutes.By detection, taken off by this method
The teeth matrix granule or freeze-dried powder fine uniform of Calcium treatment, form is consistent, and in pole shape, length is about 100-200nm, directly
Footpath is about 50-lOOnm.
2, the addition of external source bmp protein.
A, 1000 grams of addition external source bmp proteins of tooth substrate of decalcification teeth matrix processing, addition are had already passed through to step (7)
The method of foreign protein is as follows：
B, the decalcification teeth matrix first step (7) processing removes hydrogen peroxide, is rinsed repeatedly until without dioxygen with aqua sterilisa
Water is present；Obtain 100 grams of people's teeth matrix powder；
C, and then the teeth matrix powder of decalcification is allowed to be suspended in 1 liter of PH=7.2 phosphate buffer；
D, (1.0 grams, buy from Shanghai wheat bin Bioisystech Co., Ltd) PH=for using same volume of external source BMP-2
7.2 phosphate buffer dissolves, and then both mix, and is put into progress uniform speed slow stirring in whisking machines, stirring
Speed be stirred for 30-80 revs/min, while in the water-bath in alternating temperature, temperature is sequentially as follows：26 degrees Celsius, 8 is small
When；30 degrees Celsius；6 hours；28 degrees Celsius；8 hours, 28 degrees Celsius, 12 hours；Then it is maintained under 30 degrees Celsius of constant temperature and carries out
Continue to stir, every the concentration of BMP-2 albumen in 3 hours measurement mixed once solution, until concentration, to drop to initial BMP-2 dense
Below the 20%-25% of degree stops stirring, then by centrifugation, removes supernatant fluid, remaining powder is filtered, then
People's teeth matrix freeze-dried powder is obtained by low temperature drying.By detection, by this method decalcification processing teeth matrix granule or
Person's freeze-dried 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 processing addition external source bmp protein.
It is with the difference of examples of implementation 1, foreign protein BMP-7, other conditions are all.
Examples of implementation 3：People's teeth matrix carries out decalcification processing addition external source bmp protein.
It is with the difference of examples of implementation 1, while in the water-bath in constant temperature, temperature 15,20,35,38 is Celsius
Degree, time are more than 34 hours, every the concentration of BMP-2 albumen in 3 hours measurement mixed once solution, until concentration drops to
Below the 20%-25% of initial BMP-2 concentration stops stirring, then by centrifugation, removes supernatant fluid, remaining powder enters
Row filtering, then obtains people's teeth matrix by low temperature drying, is placed in 70% alcohol liquid and stores under conditions of 5 DEG C of Celsius temperature
It is standby.By detection, the teeth matrix granule or freeze-dried powder fine uniform handled by this method decalcification, form is consistent, is in
Pole shape, length are about 100-220nm, and diameter is about 40 1 11Onm.
Examples of implementation 4：2 parts of people's teeth matrixes carry out decalcification and de- albumen (BMP) processing, handle the BMP egg albumen extraction With a people's teeth matrix powder mixing that albumen processing is taken off by decalcification。
It is as follows that 1. people's teeth matrix carries out the step of decalcification processing：
(1), people's tooth of collection is placed in fresh water container, the 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 being had no value for use on tooth body, is removed；
(4) dental tissue mechanically, is ground into granule or freeze-dried powder, average grain diameter be 0.25 millimeter (such as
It can be 0.2,0.5,0.6,0.7,0.8,1.0,1.2 millimeter)；
(5) dental tissue of granule or freeze-dried powder, soaking disinfection, are ground into 1.0% sodium hypochlorite soaking disinfection again
Time is 45 minutes, and temperature is 35-40 degree Celsius；
(6), by dental tissue granule, either freeze-dried powder is placed in 0.65 (or 0.67,0.68,0.67,0.72) equivalent salt
Acid soak, soaked under 38 degrees Celsius 20 hours and complete decalcification；
(7), low temperature drying, then with 35 degree Celsius 3% of hydrogen peroxide dipping 30 minutes.
2. carry out the extraction process of bmp protein.
1000 grams of powder of tooth substrate that the processing of decalcification teeth matrix is had already passed through to step (7) extract the place of albumen
Reason, the method for extracting BMP albumen are as follows：
A, the decalcification teeth matrix first step (7) processing removes hydrogen peroxide, is rinsed repeatedly until without dioxygen with aqua sterilisa
Water is present；Obtain 1000 grams of people's teeth matrix powder；
B, and then 1000 grams of the teeth matrix powder of decalcification is allowed to be suspended in 5 liters of PH=7.2 phosphate buffer；
C, the phosphate buffer of b step is heated in water-bath 280 hours, temperature is 40 degrees Celsius, is being heated
During processing, constantly stirring；Then centrifuged, separate supernatant and deposit, supernatant is sieved with molecule
Final to obtain bmp protein activity extract from the purifying and concentration of destination protein, by measure, its molecular weight is BMP molecule
Measure as 20--50kda.Precipitation powder is filtered, dried, recovery, obtains the teeth matrix powder that 980 grams of processes take off albumen processing
End.
D, the teeth matrix powder that decalcification and de- albumen are handled and the bmp protein mixed processing of extraction are allowed, the step of processing such as
Under：The teeth matrix powder after decalcification by bmp protein extraction is allowed to be suspended in PH=7.0 phosphate buffer (100 grams first
Every liter, 5 liters of solution are configured, add up to 500 grams) and then the bmp protein (step c) phosphoric acid extracted from 1000 grams of people's teeth matrixes
Salt buffer dissolves, and then both mix, and is put into whisking machines progress uniform speed slow stirring, the speed of stirring be for
30-80 revs/min, while 12-24 hours are stirred at room temperature or under 25-30 degrees Celsius, when stirring, every
The concentration of the bmp protein of 3 hours measurement mixed once solution, until concentration drop to the 10%-15% of initial BMP concentration with
It is lower to stop stirring, then by centrifugation, remove supernatant fluid, remaining powder is filtered, then obtained by low temperature drying
Matrix, be placed under conditions of 5 DEG C of Celsius temperature in 70% alcohol liquid store it is standby.
By detection, the teeth matrix granule or freeze-dried powder fine uniform handled by this method decalcification, 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 bone carries out decalcification, de- albumen processing, then adds external source bmp protein.
It is as follows that 1. people's bone carries out the step of decalcification processing：
(1), people's bone of collection is put into fresh water container, the condition storage below 5 degrees Celsius；
(2), with 2.0% sodium hypochlorite soaking disinfection raw material bone, the time is 55 minutes, temperature be 37 degrees Celsius (or
Other temperature, such as 35,30,38,40 degrees Celsius)；
(3) part being had no value for use on bone, is removed；
(4) skeletal tissue mechanically, is ground into granule, average grain diameter, which is 0.25 millimeter, (such as can be
0.2nd, 0.5,0.6,0.7,0.8,1.0,1.2 millimeter, or the nano particle of other particle diameters, such as 50-100,120-200 nanometer
Particle)；
(5), it is ground into granule with 1.5% sodium hypochlorite soaking disinfection again, the soaking disinfection time is 45 minutes, temperature is
35-40 degrees Celsius；
(6), by skeletal tissue's granule or it is placed in the hydrochloric acid solution of 0.65 (or 0.67,0.68,0.67,0.72) equivalent
Immersion, soaked under 38 degrees Celsius 16 hours and complete decalcification；
(7), low temperature drying obtains freeze-dried powder or particle, then with 35 degree Celsius 3% of hydrogen peroxide dipping 30 minutes.
By detection, bone matter granule or freeze-dried powder fine uniform after this method decalcification processing, form is consistent, is in
Pole shape, length are about 120-240nm, and diameter is about 60 1 120nm.
2. carry out the degradation treatment of bmp protein.
Processing side to having already passed through 1000 grams of powder extraction albumen of bone matrix after decalcification bone is handled in step (7)
Method is as follows：
A, the hydrogen peroxide first in removing step (7) decalcification processing bone matrix, is rinsed repeatedly until unparalleled with aqua sterilisa
Oxygen water is present；Obtain 1000 grams of bone matrix powder；Then the bone powder of 1000 grams of decalcifications is allowed to be suspended in 2 liters of PH=7.2
Phosphate buffer in；
B, to make enzyme digestion reaction reach preferable degree, and Productive statistics cost is combined, in an experiment from pancreatin, enzymolysis
Kettle temperature is adjusted to 48-50 DEG C, and pH value is adjusted to add pancreatin O.35g when 8.5；The reaction time of enzyme is 12-56 hours, is carried out
Protein degradation processing；After processing terminates, supernatant and precipitation is centrifuged, removes supernatant, retains precipitation, and done
It is dry, obtain 900 grams of Deproteinated bone powders of decalcification.
C, and then 1000 grams of the Deproteinated bone powder of decalcification is allowed to be suspended in 5 liters of PH=7.2 phosphate buffer；
D, (1.0 grams, buy from Shanghai wheat bin Bioisystech Co., Ltd) PH=for using same volume of external source BMP-2
7.2 phosphate buffer dissolves, and then both mix, and is put into progress uniform speed slow stirring in whisking machines, stirring
Speed be stirred for 30-80 revs/min, while in the water-bath in alternating temperature, temperature is sequentially as follows：26 degrees Celsius, 8 is small
When；30 degrees Celsius；6 hours；28 degrees Celsius；8 hours, 28 degrees Celsius, 12 hours；Then it is maintained under 30 degrees Celsius of constant temperature and carries out
Continue to stir, every the concentration of BMP-2 albumen in 3 hours measurement mixed once solution, until concentration, to drop to initial BMP-2 dense
Below the 5%-6% of degree stops stirring, is then centrifuged for, removes supernatant fluid, remaining powder is filtered, then through too low
Temperature is dried to obtain bone matrix, be placed under conditions of 5 DEG C of Celsius temperature in 70% alcohol liquid store it is standby.
By detection, the bone matrix granule or freeze-dried powder fine uniform handled by this method decalcification, form one
Cause, 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 laboratory apparatus
ELISA Plate (24 holes, 96 holes), ELIASA (Model 550), electric centrifuge (80-2 types), cell culture incubator
(HeraeusBB6220 types), bidirectional magnetic force heating stirrer (Medical Instruments factory of Jintan City of Jiangsu Province), (Shanghai is accurate for acidometer
Scientific instrument Co., Ltd), thermostat water bath (Medical Instruments factory of Jintan City of Jiangsu Province), (Suzhou purifies medical superclean bench
Instrument factory.
1.2 experiment material
Material and Shenzhen light Chuan Bo biological products Development Co., Ltd from the sub- 1-5 of embodiments of the invention
Commercial teeth matrix carries out parallel control (CK) processing experiment, class standard hyclone (Hangzhou Chinese holly), DMEM culture mediums
(Sigma), 0.25% trypsase (Sigma), Tritonx-100 (Shanghai life work), ALP standard preparations box (on
Hai Shenggong).2 experimental methods
2.1MC-3T3 osteoblasts cultivation：
MC-3T3 Gegenbaur's cells are routinely recovered in the culture medium containing 10% hyclone, 50ml/L CO2, saturated humidity,
Cultivated under 37 DEG C of environment, liquid is changed after 24h, after growing to 80%, after 2.5/L Trypsin Induceds, turned generation, done with 3-4 for cell
Experiment
2.2MC-3T3 osteoblastic proliferations are studied
It is before experiment that the rich biological products development of the bright wound of the sub- 1-5 of embodiments of the invention matrix powder and Shenzhen is limited
The commercial teeth matrix powder of company weighs 0.1 gram of 24 orifice plate of addition, and every kind of hole of sample average 3, ultraviolet, which irradiates 30 minutes, to sterilize.
Cell suspending liquid is made in the cell dissociation of exponential phase, adjusts concentration, it is 1 × 10 to make inoculum density4/ ml, add 24 holes
Plate, per hole l ml, in 50ml/L CO2：Under saturated humidity, 37 DEG C of environment after culture 2,4,6d, 4 are selected successively in each culture plate
Hole, MTT liquid 160ul (microlitre) are added, continue to cultivate 4h, discard nutrient solution, add DMSO1.2ml, 10min is vibrated, by 200 μ l
(microlitre) is placed in 96 orifice plates, average three holes per sample, is determined absorbance A value in 490nm wavelength with enzyme-linked immunosorbent assay instrument, is used
Mtt assay cell count standard curve estimates cell density.Pass through single dependent variable multifactor analysis of variance pair of SPSS statistical softwares
Above experimental result is analyzed.
As a result it is as follows:
The MTT test results of different disposal are shown in Fig. 1-2.With the extension 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 control group CK, through counting credit
Analysis, the material of embodiment 1,3,4,5 is with compareing statistically significant (P between CK<O.05) (Fig. 1 and Fig. 2).It is so further
Illustrate that the skeletonization discrete phase that the present invention obtains has higher activity than commercial matrix of the prior art.With great application
Prospect.
2.3MC-3T3 cell ALP determinations of activity
Cell inoculation method is same as above, and after cultivating 2,4,6d, discards nutrient solution, PBS (0.01mol/L) is rinsed 3 times, is added
The μ l ((microlitre)) of 2ml/L Triton X 800,4 DEG C of refrigerator overnights, 300 microlitres of ALP substrates are added, 37 DEG C keep 40min,
With 0.1mol/L KOH stopped reactions, by 200 μ l, ((microlitre) moves into 96 orifice plates, average three holes per sample, with enzyme-linked immunosorbent assay instrument
Absorbance A value is determined in 410 nanometers, that is, represents Cellular alkaline phosphatase activity.To exclude proliferation rate difference to alkaline phosphatase
The influence of enzymatic activity, is corrected as follows：Gegenbaur's cell mean alkaline phosphatase activity=measure absorbance A value/same period skeletonization
Cell proliferation rate.Above experimental result is analyzed by single dependent variable multifactor analysis of variance of SPSS statistical softwares.
As a result it 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 the prior art.In addition, it also further demonstrate that BMP-2 albumen is main activated protein, for bone
Reparation or growth have the function that important.Extend with incubation time, each group Cellular alkaline phosphatase activity all increased, and implement
The alkaline phosphatase activities of example 1 and 3 is higher than control group, and through statistical analysis, examples of implementation 1 are with compareing CK, examples of implementation 3,4
And 5 respectively with compareing statistically significant (P between two groups of CK<0.05), in significant difference.So further illustrate the present invention
The skeletonization discrete phase of acquisition has higher activity than commercial matrix of the prior art.
In addition, prove (to add foreign protein and implementation in the method such as examples of implementation 1 of use by our other experiments
The foreign protein added in example 5), in all external source bmp proteins, for example, external source bmp protein is BMP-2, BMP-1, BMP-
3rd, the one or several kinds in BMP-4, BMP-5, BMP-6 or BMP-7, individually using a kind of foreign protein when, external source egg
White BMP-2, BMP-3 and BMP-7 effect are better than other albumen.
In addition, the rich biology of the bright wound of matrix granule or dry powder and Shenzhen that we obtain to examples of implementation 1,3,4,5
The commercial teeth matrix of product Development Co., Ltd carries out parallel control (CK) processing experiment, investigates for senile fracture, secondary
Fracture and three phases, the repairing effect of fourth phase caput femoris necrosis bone.As a result find, relative to the teeth matrix of existing commercial distribution
Material (into osseous granules or preparation), embodiments of the invention 1,3,4,5 obtain matrix granule or dry powder the effect of it is bright
The effect of material of the aobvious teeth matrix material, the particularly teeth matrix that examples of implementation 1,3,4 obtain for being better than current commercialization, is best, special
It is not that the effect of examples of implementation 1 and 4 is more preferable.This be probably because, existing teeth matrix material be directly over decalcification processing, wherein
The activated protein contained is natively fewer, and in acid decalcification processing procedure, active bmp protein may be caused largely
Degenerative lesion, so as to which effect is less desirable, and decalcification processing has a larger randomness, and the performance of product is with very big
Differences between batches.And the present invention can significantly improve curative effect by simply handling；In addition, from January, -2016 in January, 2014
Preserve at normal temperatures, the result for carrying out activity experiment shows, preservation under room temperature lower 2 years or so, although activity has somewhat reduced (greatly
About reduce 5-10% or so) (specific experiment process and specific data are omited), but higher activity is remained in that, store 2 years
Afterwards, activity is almost current 2-4 times of commercial product activity.This is commercially produced for a large amount of be standardized, and effect is more notable
Induced osteogenesis product provide a new approach.
Examples of implementation 7
The final products formed in of the embodiment of the present invention 1,3,4,5 add vitamin B2 with 1000 grams of amount：1
Milligram, and passes through reality by 2 milligrams, 3 milligrams, 4 milligrams, 5 milligrams, 6 milligrams, 7 milligrams, 20 milligrams, 30 milligrams, 50 milligrams, 100 milligrams
Apply 2.2 and 2.3 method that example 6 is illustrated and carry out the checking of effect, while do not add vitamin B2 as control 1, and list
Only vitamin B2 is used as with the powder mixing of inactive bmp protein and compares 2.
As a result it is as follows：
1. the MC-3T3 test results of different disposal show.
With the extension of incubation time, each group cell quantity increased.The material of embodiments of the present invention 1,3,4,5
Inoculation group cell proliferating number is higher than control 1 and control 2 after material with the addition of vitamin B2, wherein control 2 is surveyed without any substantially
The cell cultivation effect of examination.Through statistical analysis, some processing between compareing 1 with having system in the material of embodiment 1,3,4,5
Meter learns meaning (P<O.05).When the amount of addition is 1-30 milligrams, there is pole significant difference (P with compareing 1<O.01),
Make a concrete analysis of result and process is omited.Find simultaneously, different amounts of vitamin B2 is added in each processing also to be had to value-added effect
Difference, when the amount of addition is when between 1 milligram -30 milligrams, the cell proliferating number of cell can be significantly improved, and more than 30
Each processing of milligram between compareing 1 with being not significantly different, and value-added effect unobvious.
More than corresponding to each processing in the result of alkaline phosphatase activities, with the value-added effect class of Gegenbaur's cell
Seemingly.Specific experiment process is omited.This seems that proof when the vitamin B2 of certain proper ratio is added, can cooperate with BMP eggs
The white increment for carrying out Gegenbaur's cell, so further illustrate the skeletonization discrete phase of the invention obtained than commercial base of the prior art
Matter has higher activity.With great application prospect.
Claims (10)
1. a kind of method for preparing self-bone grafting osteogenic formulation, this method comprise the following steps：1. pair external source bone matrix carries out decalcification
Processing；2. and then albumen is removed to the bone matrix by decalcification processing again；3. external source bone morphogenetic protein is allowed with passing through
The bone matrix of step 2 processing is mixed, and allows foreign protein and estimate shape induced osteogenesis preparation.
2. the method according to claim 11, wherein, described bone behaviour bone, to the step of people's bone progress decalcification processing
It is rapid as follows：(1), people's bone of collection is entered in fresh water container, in less than 5 degree condition storages Celsius；(2), with 1.2%d hypochlorous acid
Sodium soaking disinfection raw material bone, the time is 30-55 minutes, temperature is 35-40 degrees Celsius Celsius；(3), remove on bone without use
The part of value；(4) skeletal tissue mechanically, is ground into granule or freeze-dried powder, average grain diameter 0.10-0.25
Millimeter；(5) skeletal tissue of granule or freeze-dried powder, soaking disinfection, are ground into 1.2% sodium hypochlorite soaking disinfection again
Time is 10-45 minutes, temperature is 35-40 degree Celsius；(6) skeletal tissue's granule or freeze-dried powder, are placed to 0.65-
0.72 equivalent hydrochloric acid solution is soaked, and 16-20 hours are soaked under 38 degrees Celsius and complete decalcification；(7), low temperature drying, decalcification is obtained
Bone matrix powder or particle.
3. method according to claim 1 or 2, wherein, the bone matrix after step decalcification bone processing is carried out
The method for extracting the processing of albumen is as follows：A, the decalcification bone matter of processing is removed hydrogen peroxide first, rinsed with aqua sterilisa multiple
Until exist without hydrogen peroxide；Obtain bone matrix powder；Then 1000 grams of the bone powder of decalcification is allowed to be suspended in 2 liters of PH=7.2
Phosphate buffer in；B, enzymolysis kettle temperature is allowed to be adjusted to 48-50 DEG C, pH value is adjusted to add pancreatin O.35g when 8.5；Enzyme
Reaction time is 12-56 hours, carries out the degradation treatment of albumen；After processing terminates, supernatant and precipitation is centrifuged,
Supernatant is removed, retains precipitation, and is dried, obtains decalcification, Deproteinated bone powder particle.
4. according to the method for claim 1, wherein, then allowing decalcification, Deproteinated bone powder is suspended in PH=7.2's
In phosphate buffer.
5. according to the method for claim 4, wherein, external source BMP PH=7.2 phosphate buffers are dissolved, then
With being mixed comprising both decalcifications, the phosphate buffer of Deproteinated bone powder, it is put into whisking machines and carries out at the uniform velocity
It is slowly stirred, the speed of stirring is stirred for 20-120 revs/min, while in the water-bath in alternating temperature.
6. according to the method for claim 5, wherein, the temperature of water-bath is sequentially as follows：26 degrees Celsius, 8 hours；30 is Celsius
Degree；6 hours；28 degrees Celsius；8 hours, 28 degrees Celsius, 12 hours；Then it is maintained under 30 degrees Celsius of constant temperature and carries out continuing to stir,
Every the concentration of bmp protein in 3 hours measurement mixed once solution, until concentration drops to the 5%-6% of initial BMP concentration
Stop stirring below.
7. according to the method for claim 6, wherein, after stopping stirring, by centrifugation, remove supernatant fluid, remaining powder
End is filtered, then by low temperature drying.
8. according to the method described in one of claim 1-7, wherein, described external source bmp protein is BMP-2, BMP-1, BMP-
One or several kinds in 3BMP-4, BMP-5, BMP-6 or BMP-7.
9. according to the method described in claim 1-8, wherein, include vitamin B2 in induced osteogenesis preparation.
10. a kind of granule or freeze-dried powder preparation for promoting bone to increase prepared such as one of claim 1-8 method, its
In, the actual granule or freeze-dried powder fine uniform, form is consistent, and in pole shape, length is about 122-240nm, diameter
About 60 1 110nm；Wherein, promote also to include vitamin B2 in the granule or freeze-dried powder preparation of bone growth.
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| CN109045359A (en) * | 2018-08-09 | 2018-12-21 | 深圳市光明创博生物制品发展有限公司 | A kind of composite bio-active material and preparation method thereof for Maxillofacial defect reparation |
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| CN111110918B (en) * | 2019-09-26 | 2020-08-21 | 中新棠国业(苏州)医疗科技有限公司 | Preparation method of high-strength implant-grade bone material |
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| US20070254040A1 (en) * | 2006-05-01 | 2007-11-01 | James Scaffidi | Nutritional supplement and use thereof |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070254040A1 (en) * | 2006-05-01 | 2007-11-01 | James Scaffidi | Nutritional supplement and use thereof |
| 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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| CN109045359A (en) * | 2018-08-09 | 2018-12-21 | 深圳市光明创博生物制品发展有限公司 | A kind of composite bio-active material and preparation method thereof for Maxillofacial defect reparation |
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